The TGF-beta-related DVR gene family in mammalian development. The genes that encode the bone morphogenetic proteins and the Vg-related proteins are mammalian members of a group of TGF-beta-related genes, designated the DVR family, that includes the decapentaplegic gene of Drosophila and the Vg1 gene of Xenopus. Members of the DVR (decapentaplegic-Vg-related) family have been implicated in diverse processes during development, particularly in epithelial-mesenchymal interactions. The results of our in situ hybridization studies with postimplantation mouse embryos provide evidence for the involvement of DVR family members, particularly DVR-2, DVR-4 and DVR-6, in specific inductive interactions during the development of many organs, including the limb, the whisker follicle and the heart.


Use of embryonic stem cells to study mutations affecting postimplantation development in the mouse. The generation and analysis of insertional mutations that perturb early postimplantation development provide a means to identify genes required at this stage of embryogenesis. We have been studying two independently generated insertional mutations termed 413.d and H beta 58 that result in early postimplantation lethality. Each mutation is associated with a distinct phenotype. 413.d mutant embryos become profoundly abnormal around the time of gastrulation: no identifiable embryonic axis or mesodermal structures are formed. H beta 58 mutant embryos proceed further in development, forming a relatively normal anteroposterior axis before developmental arrest occurs. We isolated embryonic stem cell lines homozygous for each of these mutations and assessed their differentiation abilities and developmental potential in vitro and after their introduction into wild-type blastocysts. From these studies we conclude that the 413.d mutation acts in a non-cell-autonomous fashion: mutant cells appear capable of participating, in conjunction with wild-type cells, in the formation of derivatives of all three primary cell lineages of the embryo. H beta 58 mutant embryonic stem cells are clearly pluripotent but they appear to be more restricted in their developmental potential, suggesting that the H beta 58 gene product may be required by specific tissues of the embryo.


Genetic manipulation of the mouse via gene targeting in embryonic stem cells. Gene targeting applied to totipotent embryonic stem (ES) cells is a very powerful means of creating highly specific mutations of genes in the mouse. The successful application of this technology is however constrained by both the types of mutations that can be generated at a target locus and the ability to reconstruct a germline chimera from the manipulated cells. We have developed two cell lines that can be routinely transmitted through the germline of chimeras after cloning and prolonged selection in tissue culture. We have also established a variety of methods for generating non-selected mutations at the X-linked hprt locus in ES cells. Our observations at this locus have enabled us to generate successfully a subtle mutation at the non-selectable Hox-2.6 locus.


Establishment and organization of germ layers in the gastrulating mouse embryo. By following the distribution of wheat germ agglutinin-gold-labelled cells in primitive streak stage embryos, we obtained direct evidence for a continuous recruitment of the embryonic ectoderm cells to the definitive endoderm and to the embryonic and extraembryonic mesoderm during gastrulation. The majority of the definitive endodermal cells ingressed through the anterior end of the primitive streak and were incorporated initially into the midline endoderm at the archenteron, but a small population of endodermal cells may be recruited by direct delamination from the embryonic ectoderm. The pre-existing visceral embryonic endoderm was progressively replaced, but not totally, by the newly recruited population which colonized the embryonic foregut and the notochord. The developmental fate of the recruited endoderm and that of cells in the embryonic ectoderm and the mesoderm of late primitive streak stage embryos indicate that concomitant with the establishment of the germ layers, an orderly allocation of prospective fetal tissues to specific parts of the body occurs simultaneously in all three germ layers.


The gene trap approach in embryonic stem cells: the potential for genetic screens in mice. The gene trap approach in embryonic stem cells was developed as a means to screen for genes expressed during early postimplantation development in the mouse. We have validated the approach by showing that lacZ from the integrated vector is activated by splicing to endogenous exons and expressed in embryos in patterns that mimic those of the endogenous genes. These insertions can produce developmental defects in homozygous mice. The results indicate that a large screen of gene trap cell lines on the basis of embryonic lacZ expression is feasible and should provide a new source of genes, mouse mutants and mouse strains that express lacZ in particular domains and lineages. The gene trap approach could be extended to a smaller screen for genes based on mutant phenotypes.


Clonal analysis of cell fate during gastrulation and early neurulation in the mouse. The foundation of the germ layers and the extraembryonic mesoderm from the epiblast between 6.5 and 7.5 days post coitum (p.c.) is accompanied by substantial cell proliferation. It is followed during the next 24 hours by the organization of major systems of the embryo such as the central nervous system, somites, heart and vascular system. Injection in situ of a short-term lineage label (horse radish peroxidase) into single epiblast cells at 6.7 days p.c. and analysis of the descendant clones in cultured embryos have been used to trace these processes and led to the following conclusions: (1) There is extensive but not indiscriminate cell mixing at the onset of gastrulation; epiblast cells spread towards the primitive streak and descendants are there progressively incorporated into mesoderm. (2) The fate map of the mouse epiblast at the early primitive streak stage is topologically similar to those of other vertebrates. (3) Germ layers and the extraembryonic mesoderm are not clonally distinct before gastrulation, the region of overlapping boundaries in the fate map being occupied by cells that will have descendants in more than one layer. (4) Cranial neurectoderm is mainly derived from axial epiblast immediately anterior to the primitive streak of the early streak stage embryo, clonal descendants being spread rostrocaudally in the developing neural tube. Contribution to the putative floor plate is made by progenitors some of which also contribute to notochord and mesoderm.


Use of chimeras to study gene function in mesodermal tissues during gastrulation and early organogenesis. The origin of different mesodermal tissues during gastrulation and the developmental lability of mesodermal precursors can be mapped by transplanting marked epiblast cells to the same or a different position in a host egg cylinder, and assessing the subsequent fate of transplanted tissue. This information provides the context for assessing the role of particular patterns of gene expression during mesoderm formation and differentiation. For example, the stability of Hox gene expression can be examined by transplanting transgenically marked somites that express a particular Hox gene to a position in the somite file where it is not normally expressed. Such experiments can reveal not only the cues required for Hox gene expression but also the relevance of a circumscribed pattern of Hox gene expression to a specific developmental fate. A different approach to resolving gene function is to mix mutant cells known to affect mesoderm formation with normal cells and to determine the cell autonomy of mutant cells in a normal environment. Homozygous Brachyury (T/T) embryonic stem cell lines have been isolated and injected into normal blastocysts. The presence of T/T cells in chimeras results in mesodermal defects similar to those seen in the intact mutant.


Action of the Brachyury gene in mouse embryogenesis. The murine developmental mutation T identifies a gene required in mesoderm formation. T/T mutant embryos develop normally to the primitive streak stage; during early organogenesis they show insufficient mesoderm and absence of the notochord. The mutants die at around 10 days of gestation because of the lack of the allantois. We have localized the T mutation relative to DNA markers and used a combination of genetic and molecular techniques to clone the T gene. Expression of the T gene is restricted to nascent mesoderm and to the notochord, the tissues most strongly affected by the mutation. Recent results suggest that the T gene encodes a nuclear factor involved in establishing notochord cell identity and differentiation, and is directly or indirectly involved in the organization of axial development.


Accurate estimation of glomerular filtration rate in diabetic nephropathy from age, body weight, and serum creatinine. To assess, in diabetic nephropathy, the accuracy of a method that estimates glomerular function with age, body weight, and serum creatinine as parameters. Glomerular filtration rate (GFR) was measured 57 times in 20 subjects with insulin-dependent diabetes mellitus and nephropathy with a single injection of 51Cr-EDTA. At the same time, the estimated creatinine clearance (ml/min) was calculated with the Cockroft-Gault formula (140 - age [yr]) x body wt [kg] x K/serum creatinine [mumol/L]) K = 1.23 for men, 1.05 for women These values were then corrected for body surface area (1.73 m2). For GFR measurements less than 100 ml.min-1.1.73 m-2 (n = 41), there was a strong positive correlation with the estimated creatinine clearance corrected for body surface area (r = 0.94, P less than 0.0001). The slope of this regression line did not differ significantly from identity or the y-intercept from zero. On average, the Cockroft-Gault formula (corrected for body surface area) underestimated the GFR by only 3.1 ml.min-1.1.73 m-2 (9.7 SD). This formula, corrected for body surface area, gives accurate estimates of GFR when GFR less than 100 ml.min-1.1.73 m-2. This formula could be used with an acceptable degree of confidence when repeated isotope assessments of renal function in diabetic nephropathy are impracticable.


Early sonographic evaluation for fetal growth delay and congenital malformations in pregnancies complicated by insulin-requiring diabetes. National Institute of Child Health and Human Development Diabetes in Early Pregnancy Study. It has been reported that early fetal growth retardation may be a useful marker for congenital malformations in diabetic pregnancies. To test this hypothesis, diabetic and nondiabetic women were sonographically evaluated during the first trimester. Fetal crown-rump lengths were measured sonographically at least once during the first 15 wk of pregnancy in 329 nondiabetic and 312 diabetic women. Of these, 289 nondiabetic and 269 diabetic women had sonograms before 10 wk of gestation and 283 nondiabetic and 269 diabetic women had sonograms between 10 and 15 wk of gestation. Early fetal growth delay was defined as a sonographic gestational age of greater than or equal to 6 days less than menstrual gestational age. The mean crown-rump lengths at 8 wk were 17.9 +/- 4.6 mm in the diabetic and 18.7 +/- 4.9 mm in the nondiabetic groups (P = 0.13). At 12 wk, the mean fetal crown-rump length was 58.5 +/- 8.8 mm for diabetic subjects and 60.6 +/- 8.7 mm for nondiabetic subjects (P = 0.04). Between 5 and 9 wk, 28 of 289 (9.7%) fetuses of nondiabetic subjects, 34 of 259 (13.1%) normal fetuses of diabetic subjects, and 2 of 10 (20%) malformed fetuses of diabetic subjects demonstrated growth delay (P = 0.31, normal vs. malformed diabetic). Between 10 and 15 wk of gestation, 28 of 283 (9.9%) fetuses of nondiabetic subjects, 32 of 256 (12.5%) normal fetuses of diabetic subjects, and 4 of 13 (30.8%) malformed fetuses of diabetic subjects demonstrated growth delay (P = 0.06, normal vs. malformed diabetic). Early fetal growth delay did not predict a reduced birth weight at term. Among insulin-dependent diabetic subjects who were moderately well controlled at conception, statistically significant but mild early fetal growth delay was present but did not appear to be useful clinically in predicting congenital malformations. Recommendations that growth delay demonstrated on early ultrasound be used as a predictor of congenital malformation require careful reexamination.


Nonselective loss of contrast sensitivity in visual system testing in early type I diabetes. Psychophysical methods in patients with diabetes mellitus reveal deficits of central or foveal vision. Our aim was to evaluate the contrast-sensitivity thresholds in 24 insulin-dependent (type I) diabetic patients with a short disease duration and without retinopathy, taking into account metabolic control. The control group consisted of age-matched nondiabetic subjects. None had visual or systemic symptoms. Contrast sensitivity measured at eight different spatial frequencies to sinusoidal bar patterns of 0.6-12.2 cycles/deg can detect functional defects in the spatially sensitive retinal ganglion cells or in higher visual pathways. We performed two different temporal types of contrast-sensitivity testing, dynamic (8 Hz) and static (0 Hz). Significant losses with dynamic contrast-sensitivity test at all but the highest spatial frequencies (i.e., 12.2 cycles/deg) were shown, whereas there was significant attenuation of contrast sensitivity at five spatial frequencies (1.0, 1.4, 2.2, 7.1, and 9.6 cycles/deg) in the static mode. Grating losses (less than 2SD of control means) of contrast sensitivity were found in 33.3% (dynamic) and in 72.9% (static) of eyes of diabetic patients. HbA1c values were positively correlated at variable spatial frequencies (1.0, 1.4, and 2.2 cycles/deg for dynamic test and 0.6, 1.0, 1.4, 2.2, 4.8, and 7.1 cycles/deg for static test). Our results suggest an early, generally nonselective neuronal damage of visual pathways that occurs before the onset of clinically detectable retinopathy. The visual deficit may be related directly to the effects of diabetes; repetitive minor hypoglycemic insults may contribute more than a marked hyperglycemic condition to the mechanisms underlying physiological changes along the optic nerve.


Early infant diet and risk of IDDM in blacks and whites. A matched case-control study. To investigate the role of early infant feeding in the development of insulin-dependent diabetes mellitus (IDDM) and to determine whether an association exists in both blacks and whites. Black and white diabetic subjects were recruited from the Allegheny County and Children's Hospital of Pittsburgh IDDM Registries. Extensive infant diet histories were obtained from the diabetic subjects and their nondiabetic siblings, who were used as nondiabetic control subjects. Each diabetic subject was matched outside his/her family to an unrelated nondiabetic control subject on birth order, birth year (+/- 2 yr), and race, which resulted in 211 case-control pairs with a mean birth year of 1967. In whites, diabetic subjects were less likely to have been breast-fed than control subjects (odds ratio [OR] 0.5, 95% confidence interval [CI] 0.3, 0.9). Breast-feeding prevalence did not differ between black diabetic subjects and control subjects. Duration of overall and exclusive breast-feeding did not differ between diabetic and control subjects in the black and white cohorts. The following analyses, which examined whether the timing of the first breast milk substitute to which the infant was exposed differed between diabetic and control subjects, were conducted for exposure to any breast milk substitute and to breast milk substitutes that were cow's milk based. In whites, age at exposure to any breast milk substitutes and cow's milk-based substitutes were similar between diabetic and control subjects. In blacks, the first exposure to breast milk substitutes occurred significantly earlier for any substitute (5.1 vs. 11.9 wk, P = 0.02) and marginally earlier for cow's milk-based substitutes (3.9 vs. 8.5 wk, P = 0.07) in diabetic subjects compared with control subjects. The first exposure to breast milk substitutes was more likely to occur by 3 mo of age in black diabetic subjects compared with black control subjects (OR 3.3, 95% CI 1.1-10.0) after adjusting for maternal age at birth. The addition of breast-feeding status to the model only slightly weakened this association in blacks. The analyses of this study cohort suggest that the observed protective effect of breast-feeding on the risk of IDDM may be related to differences in the age at exposure to breast milk substitutes in blacks but not in whites.


Elevated proinsulin levels related to islet cell antibodies in first-degree relatives of IDDM patients. To assess whether proinsulin levels are elevated in first-degree relatives of insulin-dependent diabetes mellitus (IDDM) patients and whether there is a relationship between proinsulin levels and the occurrence of immunological markers. Fasting proinsulin concentrations were measured in 85 first-degree relatives (54 siblings, 20 parents, 11 children) of IDDM patients and in 90 age- and weight-matched control subjects with no family history of diabetes mellitus. Fasting proinsulin levels (median, 25th, and 75th percentiles) were 8 pM (range 3.2-14 pM) in first-degree relatives and 1.7 pM (range 1.7-4 pM) in control subjects (P less than 0.0001). Proinsulin was significantly elevated in siblings (7.2 pM, range 3.8-15 pM; P less than 0.0001), parents (9.8 pM, range 6.4-13 pM; P less than 0.0001), and children (6.6 pM, range 1.8-12 pM, P = 0.04) compared with control subjects but without differences between these groups. Islet cell antibody positive (ICA+) IDDM relatives had significantly higher proinsulin levels than ICA- (16 pM; range 7.2-25 vs. 6.9 pM, range 3.1-12 pM; P = 0.02). There was no difference between individuals with and without insulin autoantibodies. No difference in proinsulin levels was observed if the relatives were subdivided according to HLA-DR sharing with the diabetic proband. Fasting proinsulin concentrations were raised not only in siblings but also in parents and children of IDDM patients. Because proinsulin is more elevated in ICA+ than in ICA- subjects, increased proinsulin levels could reflect minor beta-cell damage due to previous immunological attack.


Does glycemic control of type II diabetes suffice to control diabetic dyslipidemia? A community perspective. To assess the extent to which glycemic control by itself results in satisfactory control of diabetic dyslipidemia. A population-based case series consisting of 386 Mexican Americans and 94 non-Hispanic whites with non-insulin-dependent (type II) diabetes was studied. All subjects answered questions about their medical history and care received and underwent a standardized oral glucose tolerance test and measurements of fasting serum lipid and lipoprotein concentrations. Three definitions of dyslipidemia were used: total cholesterol greater than 6.20 mM (240 mg/dl), triglyceride greater than 2.82 mM (250 mg/dl), and high-density lipoprotein cholesterol less than 0.90 mM (35 mg/dl). Despite having removed subjects receiving lipid-lowering drugs, diabetic subjects who had been previously diagnosed and were under medical care exhibited a lower prevalence of hypertriglyceridemia than those who were newly diagnosed at the time of their survey visit, suggesting that conventional management was associated with a reduced frequency of this dyslipidemia. Among previously diagnosed cases, the prevalence of dyslipidemia rose with worsening glycemic control but there was little association with type of therapy (diet only, oral agents, or insulin) or frequency of physician visits. In general, the prevalence of dyslipidemia in diabetic subjects remained higher than in nondiabetic subjects, despite hypoglycemic therapy. The results suggest that glycemic control by itself does not suffice to control diabetic dyslipidemia and that significant numbers of diabetic subjects will need direct lipid management. Clinical trials are urgently needed to define the optimum management strategy for diabetic dyslipidemia.


Glycosylated serum protein levels assayed with highly sensitive immunoradiometric assay accurately reflect glycemic control of diabetic patients. To develop a sensitive and reliable immunoreadiometric assay to measure glycosylated lysine residues on serum proteins (GSP) and to evaluate its efficacy in monitoring glycemic control. The effect of acute and chronic in vitro and in vivo changes in glucose levels on GSP concentration was evaluated. GSP determinations from insulin-dependent diabetic (IDDM) patients, non-insulin-dependent diabetic (NIDDM) patients, and control subjects were correlated with other indices of glycemic control. The GSP levels were unaffected by acute glucose changes after food or intravenous glucose administration but increased during storage at -20 degrees C due to in vitro glycosylation by endogenous glucose. Immediate acidification of the serum prevented this, permitting long-term storage despite high ambient glucose levels. In randomly selected diabetic patients, 96% of GSP values were greater than the mean +3SD of nondiabetic control subjects. In diabetic patients, GSP levels correlated with mean plasma glucose concentrations (Kendall correlation statistics 0.47, P less than 0.001), fasting plasma glucose levels (Kendall statistics 0.42, P less than 0.001), and glycosylated hemoglobin (GHb, Kendall statistics 0.30, P less than 0.005). Induction of near-normal glycemia in poorly controlled NIDDM patients reduced GSP levels with a slope consistent with a half time of disappearance of 4.7 +/- 0.4 days. GSP levels remained elevated in 6 of 10 well-controlled NIDDM patients, despite normal GHb concentrations. Chronic hypoglycemic states, like pregnancy and hyperinsulinemic hypoglycemia, were associated with significantly low GSP levels. We describe a reproducible and sensitive immunoradiometric assay for GSP that closely reflects the degree of glycemic control in diabetic patients. Further studies are needed to determine whether this assay may be useful in screening for glucose intolerance or gestational diabetes.


In vivo demonstration of insulin-receptor defect with 123I-labeled insulin and scintigraphic scanning in severe insulin resistance. Insulin-receptor function in humans is usually studied in vitro on readily available cells, e.g., erythrocytes and fibroblasts. Although these cells are not metabolically important targets for insulin action, information derived from them are often taken as representative of other tissues. The aim of this study was to investigate insulin receptors in vitro on erythrocytes and in vivo on one of the main insulin-target organs, the liver. A 16-yr-old girl affected by severe insulin resistance was identified. Insulin receptor binding was measured on the erythrocytes of the patient and of 6 nondiabetic volunteers. The biodistribution of 123I-labeled insulin was studied in vivo by scintigraphic scanning in the insulin-resistant patient and in 10 nondiabetic volunteers. Erythrocytes of this patient displayed a markedly reduced [125I]insulin binding. In vivo 123I-insulin biodistribution was characterized by lack of hormone uptake by the liver (4 vs. 21% of the injected dose in control subjects) contrasting with intense accumulation of radioactivity in the kidneys. Our studies show that defects of insulin binding can be directly demonstrated in vivo on liver receptors with a noninvasive technique with low radiotoxicity.


Hypertriglyceridemia in different degrees of glucose intolerance in a Finnish population-based study. To determine the prevalence of hypertriglyceridemia and the mean serum triglyceride concentrations in different degrees of glucose tolerance--non-insulin-dependent diabetes mellitus (NIDDM), impaired glucose tolerance (IGT), and normal glucose tolerance (NGT). In addition, we analyzed the correlates of serum triglyceride concentration to explain why it is more prevalent in diabetic subjects. This study was a cross-sectional survey of 4000 people aged 45-64 yr randomly drawn from the population register of the Finnish population of the provinces of North Karelia and Kuopio in eastern Finland and Turku/Loimaa area in southwestern Finland and stratified by four 10-yr age- and sex groups. The final material comprised 96 subjects with NIDDM, 102 subjects with IGT, and 323 subjects with normal glucose tolerance classified on the basis of two 2-h oral glucose tolerance tests. The prevalence of hypertriglyceridemia by the glucose tolerance status and the variation in serum triglycerides associated with selected life-style and biochemical factors were executed as the main outcome measures. The prevalence of hypertriglyceridemia (greater than or equal to 2.3 mM) was 47.6% (95% confidence interval [CI] 32.5-62.7%) in NIDDM men, 21.9% (95% CI 7.6-36.2%) in IGT men, and 15.4% (95% CI 9.3-21.5%) in NGT. In women, hypertriglyceridemia was found in 51.9% (95% CI 38.6-65.2%) among those with NIDDM, 25.7% (95% CI 15.5-35.9%) among those with IGT, and 10.7% (95% CI 6.3-15.1%) in women with NGT. After adjusting for body mass index (BMI) and age, the difference in the prevalence of hypertriglyceridemia between the glucose tolerance groups remained significant in both men (P = 0.008) and women (P = 0.0001). High serum total cholesterol, high BMI, high waist-hip ratio, and low high-density lipoprotein (HDL) cholesterol were significantly associated with high serum triglycerides in all glucose tolerance groups. No synergistic effect between these parameters and glucose tolerance status was found. In multiple linear regression analyses, fasting plasma insulin, diabetes status, and serum uric acid were significant predictors of serum triglyceride concentration after taking into account age, BMI, and HDL and total cholesterol. The association between BMI and serum triglycerides in the regression analysis was significant only when plasma insulin was not included in the model. Hypertriglyceridemia is common in subjects with NIDDM and IGT and is often associated with low HDL cholesterol, high total cholesterol, hyperinsulinemia, and elevated serum uric acid concentration.


Biosynthetic human proinsulin. Review of chemistry, in vitro and in vivo receptor binding, animal and human pharmacology studies, and clinical trial experience. To describe the rationale for the preclinical and clinical developmental course of human proinsulin (HPI), the second product after human insulin for the treatment of diabetes mellitus to be manufactured by DNA technology. The relevant and available published and unpublished preclinical and clinical information generated on pork proinsulin and human proinsulin has been integrated to demonstrate how certain clinically attractive features of pork proinsulin (a soluble intermediate-acting and possibly hepatospecific insulin agonist) led to the development of HPI. Clinical pharmacology studies demonstrated that HPI was definitely, although marginally, hepatospecific. More striking was the finding that the intrasubject/patient coefficient of variation of response to HPI was significantly less than that observed with NPH insulin. However, the fact that unique efficacy in controlled multicenter studies was not demonstrated suggested that these pharmacological features were not translated into clinical benefit. In one multicenter new patient study there were six myocardial infarctions, including two deaths, in patients treated for greater than or equal to 1 yr with HPI and none in the control group. To obtain an independent review of the risks and benefits of HPI, in February 1988, Lilly convened a consultant group that examined all relevant information on HPI available. These experts shared our concerns about the safety of HPI in light of the failure to demonstrate unique efficacy. Accordingly, clinical trials with HPI were suspended in February 1988. Experience with HPI demonstrates the challenge associated with the development of new drugs in general and insulin agonists in particular.


Muscle blood flow in diabetes mellitus. Evidence of abnormality after exercise. To determine whether muscle blood flow before and after exercise is abnormal in patients with diabetes mellitus. Muscle blood flow (MBF) was measured with the 133Xe clearance technique in 15 nondiabetic subjects, 10 patients with insulin-dependent diabetes mellitus (IDDM), and 11 patients with non-insulin-dependent diabetes (NIDDM) at rest and after exercise. None of the patients had neuropathy. The median resting MBF was similar in all three groups. The median postexercise MBF was significantly greater in nondiabetic subjects (40.1 ml.min-1.100 g-1 of tissue) than in patients with IDDM (25.7 ml.min-1.100 g-1 of tissue; P less than 0.01) or NIDDM (14 ml.min-1.100 g-1 of tissue; P less than 0.01). The difference between IDDM and NIDDM was not significant. Diabetic patients have abnormalities of MBF in response to exercise. This abnormality occurs in the absence of clinical diabetic neuropathy.


Maintenance of sulfonylurea responsiveness in NIDDM. Randomized double-blind study of intermittent glyburide therapy. To assess the effectiveness of intermittent administration of sulfonylurea (glyburide) to patients with non-insulin-dependent diabetes mellitus (NIDDM). A randomized, double-blind, prospective trial compared daily administration with intermittent administration of glyburide to patients who initially responded to the drug. Twenty-eight of 60 patients with NIDDM achieved the predetermined improvement in plasma glucose concentration on glyburide therapy. These 28 responders were enrolled into a 16-wk trial of daily versus intermittent (2 wk on, 2 wk off) glyburide treatment. Laboratory assessment of glycemic control and insulin secretion in fasting and 2-h postprandial states was done every 2 wk. Patients on continuous glyburide therapy maintained their glycemic control throughout the study. In contrast, patients on the intermittent schedule lost their glycemic control immediately after being placed on placebo. Despite a significant response to each sulfonylurea pulse, these subjects never regained their baseline glycemic levels. Their fructosamine and HbA1c concentrations deteriorated and remained significantly higher than those of the continuously treated subjects. Results suggest that administration of glyburide on an intermittent basis after a 2-wk drug-free period to patients initially rendered responsive to sulfonylurea therapy is without clinical merit.


Evaluation of capillary collection system for HbA1c specimens. We evaluated the accuracy and stability of a capillary HbA1c collection system for use with a high-performance liquid chromatography analyzer. The collection system requires that 5 ul blood is drawn into a calibrated capillary tube, which is then placed into a vial of stabilizing solution and sent for analysis. The study was conducted on simultaneously drawn capillary and venous blood specimens from 47 pediatric diabetes patients. Accuracy was determined by comparing the capillary to the venous HbA1c values. Stability was measured by analyzing 17 capillary specimens over 3 wk. There was excellent agreement between the capillary and venous HbA1c values (capillary 0.959, venous +0.494, R2 = 98.7%). The capillary HbA1c values were 0.2% higher than the venous HbA1c values and decreased gradually over time (0.1% HbA1c/week) when stored at room temperature. The Bio-Rad (Richmond, CA) collection system is accurate, stable, and simple to use.


Immunological and metabolic responses of patients with history of antibody-induced beef insulin resistance to treatment with beef, pork, human, and sulfated beef insulin. We evaluated immunological and metabolic responses during therapy with beef (B), pork (P), human (H, rDNA), and sulfated beef (SB) insulins in patients with insulin-antibody-mediated insulin resistance. A randomized double-blind sequential crossover study was performed with each insulin administered for 56 days unless dose reached 200 U/day or allergy developed. Participants were 26 individuals with history of B-P insulin dosage greater than or equal to 200 U/day and insulin binding capacities greater than 0.216 nM (30 mU/ml serum). Twenty-one participants completed the study. Insulin dosage/day, fasting plasma glucose, percentage HbA1, insulin antibody binding capacity (IABC), bound insulin (BI), percentage binding of 125I-labeled B, P, and H insulins, and receptor inhibition factor (RIF) were assessed. Mean insulin dosage (U/day) was significantly greater on B (88.9) than on P (29.2), H (29.4), or SB (29.6). On B, dosage increased in 12 individuals and reached 200 U/day in 6 individuals. Mean fasting plasma glucose (12.1 mM) and HbA1 (11%) were significantly higher on B than on P, H, and SB. Mean IABC, bound insulin, RIF, and percentage of B, P, and H bound were significantly higher on B than on P, H, and SB. Prolonged treatment with SB before entry into the study (greater than 5 wk) resulted in a blunted anamnestic response to B insulin. Rechallenge with B results in anamnestic immunological response and deterioration of metabolic control. SB, H, and P insulins have equivalent effects in patients with insulin antibody-mediated immunologic resistance.


Onset of NIDDM occurs at least 4-7 yr before clinical diagnosis. To investigate duration of the period between diabetes onset and its clinical diagnosis. Two population-based groups of white patients with non-insulin-dependent diabetes (NIDDM) in the United States and Australia were studied. Prevalence of retinopathy and duration of diabetes subsequent to clinical diagnosis were determined for all subjects. Weighted linear regression was used to examine the relationship between diabetes duration and prevalence of retinopathy. Prevalence of retinopathy at clinical diagnosis of diabetes was estimated to be 20.8% in the U.S. and 9.9% in Australia and increased linearly with longer duration of diabetes. By extrapolating this linear relationship to the time when retinopathy prevalence was estimated to be zero, onset of detectable retinopathy was calculated to have occurred approximately 4-7 yr before diagnosis of NIDDM. Because other data indicate that diabetes may be present for 5 yr before retinopathy becomes evident, onset of NIDDM may occur 9-12 yr before its clinical diagnosis. These findings suggest that undiagnosed NIDDM is not a benign condition. Clinically significant morbidity is present at diagnosis and for years before diagnosis. During this preclinical period, treatment is not being offered for diabetes or its specific complications, despite the fact that reduction in hyperglycemia, hypertension, and cardiovascular risk factors is believed to benefit patients. Imprecise dating of diabetes onset also obscures investigations of the etiology of NIDDM and studies of the nature and importance of risk factors for diabetes complications.


Coronary heart disease incidence in NIDDM patients in the Helsinki Heart Study. To determine coronary heart disease (CHD) incidence among dyslipidemic subjects with non-insulin-dependent diabetes mellitus (NIDDM) and to assess the effect of lipid-modifying treatment on serum and lipoprotein lipids and the CHD incidence in these patients. Of the 4081 men participating in the Helsinki Heart Study, a coronary primary prevention trial with gemfibrozil in middle-aged men with high non-high-density lipoprotein (HDL) cholesterol (greater than 5.2 mM; 200 mg/dL), 135 had NIDDM at entry. The incidence of definite myocardial infarction and cardiac death and changes in serum and lipoprotein lipids were determined during the 5-yr trial in the NIDDM patients and compared with those observed in nondiabetic trial participants. Compared with nondiabetic subjects, NIDDM patients had lower HDL cholesterol (P less than 0.001), higher triglyceride concentration (P less than 0.0001), and greater body mass index (P less than 0.001), there were more hypertensive patients (P less than 0.001) among them. The incidence of myocardial infarction and cardiac death was significantly higher among diabetic than nondiabetic participants (7.4 vs. 3.3%, respectively, P less than 0.02). CHD incidence in the gemfibrozil-treated diabetic men (n = 59) was 3.4% compared with 10.5% in the placebo group (NS). In multivariate analysis, diabetes (P less than 0.05), age (P less than 0.0001), smoking (P less than 0.0001), low HDL cholesterol (P less than 0.05), and high low-density lipoprotein cholesterol (P less than 0.005) were independently related to CHD incidence. Gemfibrozil-induced serum and lipoprotein lipid changes in diabetic patients were similar to those observed in nondiabetic subjects. Compared with similarly dyslipidemic nondiabetic subjects, patients with NIDDM are at markedly increased risk of CHD. This elevated risk can be somewhat reduced by gemfibrozil.


Effect of glycemic control on growth velocity in children with IDDM. To determine the effect of glycemic control on growth velocity in children with insulin-dependent diabetes mellitus. One hundred twenty-two children with insulin-dependent diabetes mellitus were studied over a 5-yr period. Every 4 mo, glycemic control was assessed by measuring total glycosylated hemoglobin (GHb), pubertal status was determined by physical examination, and height was measured with a stadiometer. Height measurements were normalized for age and sex by converting them to tau scores (the number of SD above or below the mean for age and sex). Alterations in growth velocity were determined by the change in tau scores (delta tau) between visits (i.e., no change in tau score = normal growth velocity; decrease in tau score = growth deceleration; and increase in tau score = growth acceleration). A linear relationship was seen between GHb levels and the change in tau scores (r = -0.117, P = 0.001). GHb values less than 8% were associated with growth acceleration (delta tau = +0.10 +/- 0.03), and the greatest growth deceleration occurred when GHb was greater than 16% (delta tau = -0.07 +/- 0.03). The level of GHb at which growth suppression occurred (mean delta tau became negative) was dependent on pubertal status: Tanner stage 1 greater than or equal to 10%, Tanner stages 2 and 3 greater than or equal to 8%, Tanner stages 4 and 5 greater than or equal to 16%. Linear growth velocity in children with insulin-dependent diabetes mellitus is heavily related to metabolic control. Children who are prepubertal or in the early stages of puberty are the most vulnerable to growth suppression. Once puberty is well established, growth suppression does not occur until marked hyperglycemia (GHb greater than 16%) exists.


Morning or bedtime NPH insulin combined with sulfonylurea in treatment of NIDDM. To compare the effect of morning and bedtime NPH insulin combined with daytime sulfonylurea on glycemic control in non-insulin-dependent diabetes mellitus (NIDDM) patients no longer responding to treatment with sulfonylureas alone. Twenty-four NIDDM patients who fulfilled these criteria were randomized to treatment with Protaphan human insulin in the morning or at bedtime (22 +/- 1 IU) plus 3.5 mg glibenclamide twice a day. Morning and bedtime NPH insulin resulted in equal reduction of HbA1 (from 13.5 +/- 0.3 to 9.4 +/- 0.1 and 9.6 +/- 0.2%, respectively) and mean self-monitored blood glucose (9.2 +/- 0.5 vs. 10.1 +/- 0.4 mM). Bedtime insulin resulted in lower morning blood glucose (7.8 +/- 0.5 vs. 9.1 +/- 0.4 mM; P less than 0.01), whereas morning insulin resulted in lower evening blood glucose (10.1 +/- 0.6 vs 12.1 +/- 0.6 mM, P less than 0.01). Morning and bedtime NPH insulin combined with glibenclamide are equipotent in the treatment of NIDDM patients with secondary failure to sulfonylurea. However, this treatment regimen normalizes blood glucose only in a small group of patients. Therefore, more intensified insulin therapy seems to be required to achieve this goal.


Intracellular free magnesium deficiency plays a key role in increased platelet reactivity in type II diabetes mellitus. Mg deficiency may be an important factor leading to cardiovascular disease. Diabetic subjects show an increase in platelet reactivity that can enhance the risks of vascular disease. In addition, diabetic patients have been reported to be at risk of developing extracellular Mg deficiency. However, the intracellular free Mg concentration and its role in the enhanced platelet reactivity in diabetes is not known. We evaluated the intracellular erythrocyte (RBC) Mg2+ concentration in 20 non-insulin-dependent (type II) diabetics. In addition, the effects of intravenous 3-h drip or 8 wk of oral Mg supplementation on intracellular RBC Mg2+ levels and platelet reactivity was studied. To more clearly evaluate the direct role of Mg in these effects, we induced isolated Mg deficiency in 16 nondiabetic control subjects with an Mg-free liquid diet for 3 wk. The intracellular RBC Mg2+ concentration of diabetic patients was significantly reduced compared with values in nondiabetic control subjects (166 +/- 7 vs. 204 +/- 7 microM, P less than 0.01). Serum Mg levels were also reduced in the diabetic patients compared with the control subjects (1.59 +/- 0.04 vs. 1.9 +/- 0.1 mEq/L, P less than 0.05). Oral Mg supplementation for 8 wk (400 mg/day) restored RBC Mg2+ concentration to normal without significantly changing serum Mg concentration. Both intravenous and oral Mg supplementation markedly reduced platelet reactivity in response to the thromboxane A2 analog, U46619. The Mg-free diet resulted in a significant reduction in RBC Mg2+ concentration and markedly enhanced the sensitivity of platelet aggregation to U46619 and ADP. These results suggest that type II diabetic patients have intracellular Mg2+ deficiency and that Mg deficiency may be a key factor in leading to enhanced platelet reactivity in type II diabetes. Therefore, Mg supplementation may provide a new therapeutic approach to reducing vascular disease in patients with diabetes.


Stress buffering and glycemic control. The role of coping styles. To test the hypotheses that chronic psychosocial stress is associated with worse glycemic control and that coping moderates (buffers) this effect. Subjects consisted of 105 insulin-treated adults from the Diabetes Division of Henry Ford Hospital who filled out questionnaires on stress and coping and received an HbA1 test at a clinic appointment. Six coping styles were examined, including both emotion- and problem-focused styles. Two standardized stress inventories were administered. Ineffective coping was defined as scoring below the median for stress-dampening coping styles and above the median for stress-exacerbating styles. Stress was significantly (P less than 0.05) correlated with higher HbA1 in all but one ineffective coping subgroup. Conversely, none of 12 correlations between stress and glycemic control was significant in the effective coping subgroups. Chronic psychosocial stress is associated with worse glycemic control among those who do not cope effectively with stress. Effective coping can protect individuals from the deleterious effects of stress.


Sensory function at diagnosis and in early stages of NIDDM in patients detected through screening. We studied whether sensory function abnormalities are present at diagnosis and whether they develop in the early stages of non-insulin-dependent diabetes (NIDDM). Quantitative assessments of vibration sensitivity at the hallux and index fingers, and thermal sensitivity at the hallux were performed at screening (2-h oral glucose tolerance tests) for diabetes in 364 individuals. Twenty-five subjects diagnosed with NIDDM and 25 matched nondiabetic subjects were restudied after an interval of 12-41 mo. When those with NIDDM (n = 41) or impaired glucose tolerance (IGT) (n = 38) were compared with nondiabetic subjects, there were no significant differences in sensory function indices. However, the vibration threshold and HbA1c were related among those found to be hyperglycemic (IGT and NIDDM combined P less than 0.05; NIDDM alone P less than 0.05). Among diabetic subjects, the vibration threshold and interaction term of height and Hba1c were positively related in a multiple regression analysis (P less than 0.01). There were increments in all sensory thresholds in diabetic patients at follow-up (P less than 0.05 for all). Increments were smaller in control subjects. These data suggest that although sensory function tends to be normal at diagnosis in NIDDM patients, there appears to be a diminution in sensory function as the disease progresses. An interaction between metabolic factors and height may influence sensory function early in the course of NIDDM.


Effect of thermal biofeedback-assisted relaxation training on blood circulation in the lower extremities of a population with diabetes. Investigate the effect of relaxation training/thermal biofeedback on blood circulation in the lower extremities of diabetic subjects. Diabetic subjects (n = 40) aged 17-73 yr were volunteers recruited through the University of Wisconsin-La Crosse, the local ADA Chapter, and a medical clinic. A within-subjects experimental design was used. During phase 1, all subjects used a self-selected relaxation method and recorded toe temperatures daily. During phase 2, subjects were taught a biofeedback-assisted relaxation technique designed to elicit sensations of warmth in the lower extremities and increase circulation and temperature. Subjects relaxed at home with the use of a designated relaxation tape. They measured and recorded toe temperatures. Each phase lasted 4 wk. Toe temperature and blood volume pulse (BVP) data were gathered at the beginning and end of phases 1 and 2. Paired t tests compared the means of temperature percent change scores between 1 and 2. Mean temperature change scores were 8.73% (phase 1) and 31.38% (phase 2) (t = -8.00, df = 39, P less than 0.001). Mean BVP change scores were 2.33% (phase 1) and 22.47% (phase 2) (t = -9.24, df = 35, P less than 0.001). Based on eta squared, 71% of the BVP increase in phase 2 was attributed to the relaxation technique. A multiple regression analysis indicated that none of the other examined variables affected by diabetes were significant predictors of BVP increase. Data indicate that diabetic patients show significant increases in peripheral blood circulation with this technique. This noninvasive method could serve as an adjunct treatment for limited blood flow in some complications of diabetes.


Controversial beliefs about diabetes and its care. The purpose of this study was to identify specific beliefs that differentiate health-care professionals whose attitudes toward diabetes agreed most strongly with a group of national diabetes experts from those whose attitudes disagreed most strongly. The sample for this study included 271 physicians, 834 nurses, and 546 dietitians who completed a Diabetes Attitudes Survey. The sample included specialists in diabetes care and nonspecialists. Controversial beliefs about diabetes and its care were determined by comparing the beliefs of the 10% of the sample whose attitudes were most concordant (with the national panel) with the beliefs of the 10% of the sample whose attitudes were the most discordant. Ten beliefs met the criteria for being defined as controversial. The most controversial beliefs concerned whether the patient or the physician should be the primary decision maker in diabetes care, the meaning of patient noncompliance, and the seriousness of non-insulin-dependent diabetes mellitus. The 10% of the sample with the most discordant attitudes contained a disproportionately large number of physicians, nonspecialists in diabetes, and health-care professionals who had been in practice longer than the other members of the sample. This study identifies some important differences in beliefs between younger health-care professionals who specialize in diabetes and older nonspecialists. Such beliefs should be addressed in continuing education programs with the aim being to foster the widespread adoption of a contemporary approach to diabetes care.


Metabolic impact of education in NIDDM.

Metabolic impact of education in NIDDM. To evaluate the impact of a model program of diabetes education and weight reduction on diabetes control and weight loss in obese individuals with non-insulin-dependent diabetes mellitus (NIDDM). Eighty-two obese diabetic subjects were randomized to three levels of educational intensity: 1) a single individual session, 2) a 12-wk behavior-oriented diabetes education and weight control group intervention, or 3) group intervention plus six individual follow-up sessions. Repeated measures of weight, fasting blood glucose, and HbA1c were collected. Measures of diabetes knowledge, skills, and attitudes were also obtained. By 6 mo, all three intervention groups had a significant weight loss (P less than 0.01). The mean weight loss of approximately 10 lb was independent of treatment group and was maintained over the duration of the study. However, significant improvement in metabolic control was associated with participation in the diabetes education-weight reduction intervention. The findings of this study indicate that a cognitive behavioral group intervention of diabetes knowledge and weight reduction training can produce weight loss and improvements in diabetes control. The addition of individual counseling as a follow-up maintenance strategy does not appear to have any advantage.


Use of health maintenance organization data bases to study pharmacy resource usage in diabetes mellitus. To evaluate use of medications by patients with diabetes. We analyzed the computerized pharmacy data base of a large health maintenance organization to evaluate cost impact and patterns of usage for all pharmacy products by a defined population of diabetic patients. Diabetic patients received 31.2 dispenses/yr compared with 11.5 for control patients. Total pharmacy costs were threefold higher for diabetic patients ($621 vs. 207/yr) and accounted for approximately 17% of their total health-care costs. Products specifically for the care of diabetes averaged $175/yr or approximately 4.5% of their total health-care costs: 57% received an oral hypoglycemic agent, 34% received insulin, and 6% received both. Cardiovascular drugs accounted for 29% of total costs. There was increased use of most classes of medication by patients with diabetes. Health maintenance organization management data bases, particularly those storing pharmacy dispensing data, provide useful information on the impact of chronic disease. People with diabetes receive a greater number of most types of medication with a greater overall cost than do nondiabetic people. Previous studies have underestimated the contribution of pharmacy purchases to the costs of caring for a diabetic population.


Clinical trial of programmable implantable insulin pump for type I diabetes. The first step in the evolution of an artificial pancreas is the development of a reliable implantable pump for insulin delivery. Despite recent advances, significant issues remain, including small size of studies and frequent irreversible catheter obstructions. We report safety, feasibility, and efficacy results from 56 patients, representing 73 patient-yr of pump experience, entered into a multicenter trial with a new implantable programmable pump. All patients had insulin-dependent (type 1) diabetes, were 38 +/- 8 yr old, and were not prone to severe hypoglycemia. The pump (Infusaid 1000) has a pulsatile mechanism powered by freon-vapor pressure. Its rate is regulated by battery-powered valves, operated via a hand-held programmer. The pump is refilled transcutaneously with 25 ml U100 insulin (Hoechst 21PH) on a monthly basis and has a second septum (side port) proximal to the catheter, which allows flushing the catheter or lavaging the pump unit. The pumps were implanted after 3 mo intensive subcutaneous insulin therapy and catheters were positioned either in the peritoneum (i.p., n = 38) or the superior vena cava (i.v., n = 18). All implanted pumps have functioned safely with no instance of overdelivery or stoppage. The most frequent complications were flow slow downs, presumably due to insulin precipitation within the pump, which occurred in 86% of pumps and were resolved in all but one case by lavaging the pump in situ with alkaline solution. Flow slow downs due to catheter obstruction occurred in 52% of the intravenous catheters but only 21% of the intraperitoneal catheters (P less than 0.05) and were resolved in all but two cases by diluent flushing through the sideport. Incidence of severe hypoglycemia decreased from 0.47 before implant to 0.05 episodes/patient-yr after pump implantation (P less than 0.001). Mean HbA1c fell from 7.4 +/- 1.2% after intensive subcutaneous therapy to 7.1 +/- 1.0% 12 mo after implantation. Only 2 patients withdrew from study after recurrent catheter problems, and quality-of-life questionnaires showed improvement in satisfaction with diabetes-specific quality of life when on implantable pump therapy. Insulin therapy with implantable pumps is effective and safe for periods up to 1.7 yr with a decreased risk of severe hypoglycemia than with intensive subcutaneous insulin therapy.


Lipid and lipoprotein levels in patients with IDDM diabetes control and complication. Trial experience. The DCCT Research Group. To compare lipid and lipoprotein levels in patients with insulin-dependent diabetes mellitus (IDDM) with values in similarly aged nondiabetic subjects and analyze the effects of demographic variables, metabolic control, and other factors on the lipid and lipoprotein levels in the diabetic population. Fasting total cholesterol, triglyceride, low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol levels measured during screening for the Diabetes Control and Complications Trial (DCCT) in 1569 generally healthy IDDM patients, between the ages of 13 and 40 yr and with 1-15 yr diabetes duration, were compared with values obtained with similar methods in the Lipid Research Clinics (LRC) prevalence study of nondiabetic individuals. When similarly aged groups of IDDM and nondiabetic subjects were compared, higher total and LDL cholesterol and lower HDL cholesterol lipid profiles were restricted to younger (13-24 yr) female diabetic subjects. In general, IDDM males and older IDDM females had similar or slightly less atherogenic profiles than similarly aged nondiabetic subjects. Metabolic control, measured by HbA1c, and body weight (% of ideal body wt) correlated positively with total cholesterol, LDL cholesterol, and triglyceride levels. Lipid and lipoprotein levels in the generally healthy IDDM volunteers for the DCCT are similar to those in the nondiabetic population. More atherogenic profiles were present only in younger IDDM women compared with nondiabetic women. The differences between the DCCT data and older studies that demonstrated more profound abnormalities in lipid levels in IDDM populations may reflect patient selection and/or changes in dietary patterns (higher-carbohydrate and lower-fat content) and in glucose control that has occurred in recent years.


Temporal variation in incidence of IDDM in Canterbury, New Zealand. To establish the statistical significance of observed variations over the last decade in the incidence of insulin-dependent diabetes mellitus (IDDM) in the 0- to 19-yr-old age-group and to determine whether incidence has increased in Canterbury, New Zealand. The Canterbury, New Zealand, Diabetes Registry has recorded all incidence cases of diabetes mellitus prospectively since 1982. All IDDM subjects aged 0-19 yr at diagnosis and using insulin are included in the study. Ascertainment is believed to be 100%. Prevalence was recorded at 1 January 1982 and 1 January 1990. Annual incidence for 1982-1990 was determined using age and sex cross-sectional census population denominators. The statistical significance of temporal, age, sex, and seasonal variations in incidence rates was ascertained by Poisson regression models (GLIM statistical software). Prevalence on 1 January 1990 was 115/100,000. Incidence rates during the 9 yr were periodic, with two major peaks--one in the early 1980s, the other in 1989 continuing into 1990. The temporal variation (P less than 0.02) was not age or sex specific. Incidence rates for boys were three- to fourfold higher during peak versus trough years, with a peak level of 20.7/100,000 in 1990. For girls, there was less variation, with a peak rate of 21.6/100,000 in 1990. There has been no significant increase in IDDM incidence over time. The mean rate of incidence across all age-groups for 1982-1990 was 12.7/100,000 person-yr. A significant seasonal association to the onset of IDDM was found only in boys, with incidence rates being significantly higher in winter than in summer (P less than 0.01). IDDM in Canterbury, New Zealand, presents in cycles of incidence peaks and troughs, each spanning 2-3 yr.


Xerostomia in diabetes mellitus.

Xerostomia in diabetes mellitus. To determine the prevalence of xerostomia in a group of ambulatory diabetic patients and to compare the following in patients with and without xerostomia: 1) flow rates of saliva and lacrimal fluid, 2) the presence of other symptoms suggestive of oral and extraoral dryness, 3) indexes of glycemic control, and 4) noninvasive measures of cardiovagal autonomic nervous system function. Forty adult diabetic patients and an equal number of age- and sex-matched healthy nondiabetic control subjects were studied. Subjects who consumed xerogenic drugs or had other significant diseases were excluded from the study. A questionnaire was administered to all patients, and the following tests were performed: resting and stimulated flow rates on whole saliva; Schirmer's test (lacrimal fluid), serum glucose and HbA1, expiration-inspiration ratio, 30:15 ratio, Valsalva ratio, and the systolic blood pressure response to standing. Forty-three percent of diabetic patients complained of xerostomia, of which 82% were women. The oral dryness was not related to age or the type and duration of diabetes. Symptoms of water loss and oropharyngeal, ocular, and vaginal dryness were much more common in the xerostomic than the nonxerostomic diabetic patients. The salivary flow rates of the diabetic subjects was consistently lower than those of healthy, nondiabetic control subjects. The mean, resting, and whole-saliva flow rate was abnormally low in the diabetic patients who complained of xerostomia; no significant differences were observed for the stimulated salivary and the lacrimal flow rates. Significant inverse relationships were shown between salivary flow and the level of HbA1; none were shown between flow and autonomic function. Dry mouth is a common complaint among ambulatory diabetic patients. It is strongly associated with objective measurements of poor salivary flow and with other oral and extraoral symptoms of desiccation. The oral dryness is not associated with cardiovagal autonomic system dysfunction but may be due to disturbances in glycemic control.


Foot pressure measurements in diabetic and nondiabetic amputees. Foot problems are common in the remaining foot of diabetic amputees. Because high foot pressures are associated with foot ulceration, we studied foot pressures of the remaining foot of diabetic and nondiabetic unilateral amputees. Four age-matched groups of 11 subjects were studied. The groups were comprised of diabetic subjects with previous major amputation, nondiabetic nonneuropathic amputees, diabetic nonamputee patients with similar peripheral nerve involvement as the diabetic amputees, and nondiabetic control subjects. Vibration perception threshold (VPT) was assessed by biothesiometry and foot pressures with an optical pedobarograph. Mean +/- SE VPT in the diabetic amputees was significantly higher than the nondiabetic amputees (40.2 +/- 3.7 vs. 17.7 +/- 2.8 V, P less than 0.002) and similar to diabetic nonamputees (43.4 +/- 3.3 V, NS). VPT was abnormal in 9 (82%) diabetic amputees, 2 (18%) nondiabetic amputees, and 10 (91%) nonamputee diabetic patients. The mean peak foot pressure in the diabetic amputees was higher compared with nondiabetic amputees (18.3 +/- 2.2 vs. 11.3 +/- 1.4 kg/cm2, P less than 0.05) and control subjects (10.0 +/- 1.5 kg/m2, P less than 0.01), but no difference existed with diabetic nonamputees. Abnormally high foot pressures (greater than 12.3 kg/cm2) were present in 8 (73%) diabetic amputees, 3 (27%) nondiabetic amputees, 8 (73%) diabetic nonamputees, and 4 (36%) healthy subjects. We conclude that high pressures are present under the remaining foot in diabetic amputees, and that these pressures are associated with diabetic neuropathy. Prosthetic usage does not increase the pressures under the remaining foot in nondiabetic amputees.


Prevalence of cardiovascular autonomic dysfunction assessed by spectral analysis and standard tests of heart-rate variation in newly diagnosed IDDM patients. To determine the prevalence of cardiovascular autonomic nerve dysfunction in patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM) compared with healthy nondiabetic subjects. A battery of cardiovascular reflex tests was performed in 130 newly diagnosed IDDM patients aged 12-40 yr at mean blood glucose levels of 7.2 mM after insulin had been administered for 3-39 days. Age-dependent lower limits of normal of these tests were defined at the 2.3 percentile in 120 nondiabetic subjects. Tests of heart-rate variation (HRV) included the coefficient of variation (C.V.) and the low-frequency (LF), midfrequency (MF), and high-frequency (HF) bands of spectral analysis at rest, HRV during deep breathing (C.V., expiratory-inspiratory ratio, and mean circular resultant), Valsalva ratio, and maximum/minimum 30:15 ratio. In addition, spectral analysis on standing, the change in systolic blood pressure to standing, and diastolic blood pressure response to sustained handgrip were determined in 50 patients. A significantly higher percentage of abnormal test responses in the diabetic group compared with the control group was noted for power spectrum in the LF band (7.3 vs. 0.8%, P less than 0.05) and MF band (10.6 vs. 0%, P less than 0.001) at rest and HF band on standing (10.0 vs. 0.9%, P less than 0.05), maximum/minimum 30:15 ratio (25.4 vs. 5.0%, P less than 0.001), and Valsalva ratio (17.5 vs. 4.2%, P less than 0.001). There were no significant differences between both groups in regard to the remaining parameters. Ten (7.7%) diabetic patients but none of the nondiabetic subjects had cardiovascular autonomic neuropathy defined by the strict criterion of abnormal results in more than three of six tests (P less than 0.001). In addition, 12 (9.2%) patients but only 2 (1.7%) control subjects had abnormal results in two of six tests (P less than 0.01). Cardiovascular autonomic nerve dysfunction is relatively common in newly diagnosed IDDM patients after correction of the initial metabolic imbalance. A combination of tests based on spectral and conventional analysis of HRV appears suitable for detection of early abnormalities in autonomic function in diabetes.


Association of waist-hip ratio with diabetes mellitus. Strength and possible modifiers. To characterize the association between central obesity, as measured by the waist-hip ratio (WHR), and non-insulin-dependent diabetes mellitus (NIDDM), considering the effects of sex, age, overall obesity, and family history of diabetes. Case-control study nested within a community-based survey. We selected 151 subjects with NIDDM and 301 nondiabetic control subjects as a systematic sample of survey screening negative individuals. Odds ratios for NIDDM, comparing a high WHR (greater than or equal to 0.926 for men, greater than or equal to 0.83 for women) to a low WHR were 4.72 with a 95% confidence interval of 2.39-9.34, and 2.17 with a 95% confidence interval of 1.03-4.58, for women and men, respectively, controlling for age, overall obesity, and a family history of diabetes. Women with high WHRs in the presence of these risk factors are notably at risk for diabetes. Central obesity, as measured by the WHR, is importantly and independently associated with NIDDM.


Low incidence of childhood IDDM in district of Novosibirsk (Russia). To study the incidence of insulin-dependent diabetes mellitus (IDDM) among 0- to 14-yr-old children of the district of Novosibirsk, in western Siberia, Russia, from 1983 to 1989. Data were collected with methods recommended by the Diabetes Epidemiology Research International Group. The average yearly incidence among males and females was 4.7 (95% confidence interval 3.9-5.8) and 4.4 (95% confidence interval 3.6-5.4) per 100,000 people, respectively. The incidence of IDDM among children living in the city of Novosibirsk was significantly higher than in children living in the surrounding counties of the district, 5.7/100,000 (95% confidence interval 4.8-6.8) versus 3.6/100,000 (95% confidence interval 2.9-4.5). The incidence of childhood IDDM among Russians in Siberia is low. This is the first publication of internationally comparable data on the occurrence of childhood IDDM in the Russian population.


[Myocardial contrast echocardiography for the follow-up after percutaneous transluminal coronary angioplasty]. Myocardial contrast echocardiography was performed, before and after successful elective percutaneous transluminal angioplasty (PTCA) of a main coronary artery, in 35 patients (31 men, 4 women; mean age 56 +/- 6 years). After intracoronary injection of microbubbles-containing 2 ml iopromide, contrast half-life (t/2) and maximal echo-intensity (Imax) in the myocardial region supplied by the target vessel were measured. While t/2 decreased from 8.3 +/- 5.4 s to 5.7 +/- 3.3 s (P less than 0.0002), mean Imax remained unchanged (27.4 +/- 10.7 vs 26.1 +/- 10.1 grey value units). Repeat cardiac catheterization with contrast echocardiography was performed a mean of 37 weeks (7-53) later in 13 of 35 patients with optimal echo image quality. Re-stenosis of at least 75% was demonstrated in six patients, while in seven vessel diameter had decreased by less than 30%. In all patients with re-stenosis t/2 had increased by about 20 to 100% of the initial value. It was always over 5 s (mean 6.2 +/- 1.6 s before, 4.7 +/- 1.7 after PTCA, and 6.2 +/- 1.2 s at the end of the follow-up). In one patient a prolonged t/2 persisted due to vessel dissection. Mean t/2 remained unchanged in patients without re-stenosis (5.6 +/- 2.1 s before, 3.5 +/- 1.15 s immediately after PTCA, and 3.6 +/- 1.25 s at the follow-up examination). These data suggest that contrast half-life is suitable for demonstrating changes in myocardial perfusion after PTCA.


[The piezoelectric lithotripsy of gallstones. The acute- and long-term results]. Extracorporeal piezoelectric lithotripsy (PEL) with oral lysis (about 7.5 mg/kg urso- and chenodeoxycholic acid as single dose in the evening) was performed, according to a standardized treatment and follow-up protocol, in 219 patients (177 women, 42 men; aged 47 +/- 14 years) with symptoms of gallbladder stones. The average number of treatment sessions per patient was 2.0 +/- 0.8. Significantly fewer sessions with fewer shockwave charges were required in solitary gallstones of less than or equal to 20 mm diameter than in those of greater than 20 mm diameter and in multiple concrements (P less than 0.01). Fragmentation was successful in 99% of patients. Sedation and/or analgesia during PEL were required in only 2% of patients. There were no marked side effects during the treatment. The stone-free rate 12 months after the start of treatment was 76% in the group with solitary stones less than or equal to 20 mm, 75% with solitary stones greater than 20 mm and 64% for multiple stones. During the follow-up period 36% of patients had biliary colics and 3% had fragments impacted in the common bile duct. Biliary pancreatitis occurred in 1% of patients. PEL is an effective and sparing procedure in the treatment of selected patients with gallbladder stones.


[Acute aortic insufficiency following endocarditis due to infection with Campylobacter fetus subspecies fetus]. A 50-year-old male alcoholic addict, examined because of diarrhoea with fever was found to have Campylobacter jejuni in blood and stool cultures. After administration of broad-spectrum penicillin all acute symptoms disappeared but he lost 8 kg within 3 months and his general state health gradually deteriorated. After 3 months he suddenly developed leg oedema, dyspnoea and bouts of fever up to 38.8 degrees C. A loud cardiac murmur was now heard. Echocardiography demonstrated vegetations on the regurgitant aortic valve. Endocarditis being suspected he was at first treated with penicillin G (15 mega IU/d) and gentamycin (160 mg/d). The fever regressed, but after 8 days the blood culture grew Campylobacter fetus subspecies fetus. Antibiotic treatment was switched to imipenem, twice daily 500 mg, in accordance with sensitivity test results. Further blood cultures were sterile. Despite this the cardiac status deteriorated, the aortic regurgitation reaching grade IV. The valve was replaced with a bioprosthesis and the patient quickly improved postoperatively. Antibiotic treatment was stopped and the cardiovascular status became normal. The patient has now been free of symptoms and recurrence for 7 months.


[Severe tetraparesis as the first manifestation of a chronic lymphatic leukemia]. Uncertain gait and weakness on standing, progressing to flaccid paraparesis of both legs, developed in a 76-year-old woman. Proprioceptor reflexes of both legs were absent and there was pallaesthesia up to the pelvic rim. Electromyography pointed to polyneuritis. Administration of prednisone, 1 mg/kg daily for 10 days, was ineffective, as was one-time plasmapheresis. An incomplete tetraparesis developed. Blood white-cell count increased within 2 weeks by 10,400/microliters (52% lymphocytes) to 17,400/microliters. Blood smear, bone-marrow cytology and histology, as well as immunocytology, revealed lymphoplasmocytoid immunocytomas (chronic lymphocytic leukemia). Oral cytoreductive treatment was started with chlorambucil, 0.1 mg/kg daily, and prednisone, 100 mg daily. After 19 days the patient was able to walk with support, after 28 was walking unaided. Treatment was continued over eight cycles (14 days' treatment, 14 days' interval per cycle). 17 weeks after onset of treatment paraesthesias of the legs required renewed administration of chlorambucil, 0.1 mg/kg per day, for 3 months. The patient has now been free of symptoms during a follow-up period of 12 months.


[The effect of heavy metals on the in vitro interaction between human sperm and cervical mucus]. The sperm-cervical mucus-penetration test (SCMPT) was used in vitro to examine the influence of heavy metals (mercury, cadmium and copper) on sperm function. 30 each of freshly obtained different sperm samples with varying starting sperm motility and oestrogen-stimulated cervical mucosa from women of known fertility were used. Mercury chloride (HgCl2; 0.005-0.5 mmol/l), cadmium chloride (CdCl2; 0.05-10 mmol/l) and copper sulphate (CuSO4; 0.0001-10 mmol/l) were added to the in-vitro system. After incubation, at 37 degrees C for 6 hours, linear progression, penetration depth and motility index of the sperm (cumulative SCMPT score) were analysed. An untreated control was run in parallel with each test. The added substances, especially CdCl2 and HgCl2, significantly reduced the quantitative and qualitative penetrability of sperm (P = 0.001), in step with the concentration. The concentrations which were used, especially those of mercury, were in the range of in-vivo. measured levels. These results make clear that heavy metals can influence human sperm function.


[Cholesterol--a marker for multiple risk factors? A comparison between coronary disease and coronary health]. Comparison was made in a cross-sectional study between 658 patients in whom coronary arteriography had shown (n = 304) or excluded coronary artery disease (CHD) (n = 354) and a clinically healthy group as controls (n = 1658), to assess possible risk factors. Patients aged 31-40 years with CHD had the highest total cholesterol levels (286 +/- 43 mg/dl) and the highest number of risk factors (3.6 +/- 0.9) compared to patients without CHD of the same age (204 +/- 30 mg/dl; 2.0 +/- 0.5) and healthy controls (216 +/- 45 mg/dl; 1.7 +/- 0.5) (P less than 0.001). In older patients with CHD, total cholesterol levels were lower (age group 61-70 years: 231 +/- 49 mg/dl), reaching about the same level as that of patients without CHD (232 +/- 54 mg/dl) or healthy controls of the same age (228 +/- 55 mg/dl). Furthermore, it was demonstrated that increased total cholesterol concentration can indicate the presence of other risk factors. It would thus appear that the level of total cholesterol in patients with CHD is decisively influenced by age and the presence of other risk factors.


[Pacemaker implantation via a persistent left superior vena cava in atresia of the true superior vena cava]. Implantation of a single-chamber pacemaker was planned in an 83-year-old woman with sick-sinus syndrome causing dizziness, bradycardia and tachycardia. After puncture of the right subclavian vein it proved impossible to advance a guide-wire into the superior vena cava, under fluoroscopy the wire always being seen to coil in the left subclavian vein and hence passing into a caudally directed vein. This vessel proved to be a persistent left superior vena cava (PLSVC) which connected to a markedly dilated coronary sinus (2 cm diameter) opening into the right atrium. An 85-cm electrode was then passed via the PLSVC and right atrium into the right ventricle without difficulty and was anchored in its apex. Echocardiography failed to reveal any further anomaly. Three-dimensional computed tomographic reconstruction established atresia or agenesis of the (right) superior vena cava. Normal pacemaker function freed the patient of all symptoms postoperatively.


[Veterinary medicine and environmental hygiene--20 years of seminars on environmental hygiene]. The changes of the environment after industrialization since 1965 influenced some political decisions concerning a critical evaluation of such development which may injure the environment. The School of Veterinary Medicine Hannover discussed in that time how the problems could be met by establishing a postgraduate stadium of environmental sciences. These ideas were realized, starting with seminars in the first year for one week, later for one or two days periods (Table). Postgraduate students and interested veterinarians with questions arising from their occupation participated in the seminars. The main topics of the seminars referred to effects of chemical, physical and biological influences from the environment upon farm animals and vice versa that part of animal housing which is involved in the harmful changes of the environment.


[Environmental hygiene tasks of the veterinarian in practice]. Apart from his direct tasks like treating diseased animals, preservation of animal health, recognition of mal-feeding and failures, recognition of unsuitable keeping systems and acknowledgement of animal welfare the veterinary practitioner can be the environmental-hygienic consultant of the farmer in following areas in particular: management and technique of feeding; management and technique of water supply; management and technique of air quality and ventilation; management and technique of manure handling and hygiene; cleaning and disinfection; deratting and pest control. The aim of these tasks is to diminish the risks/hazards for food products (food control act), the air (emission control act), surface water (law on the water household), ground water (waste control act, law on the water household) and vegetation and soil (waste control act, nature conservation act). Some examples will be given and comments are made on how to run a practice in an environmentally friendly way.


[Tasks of public veterinary affairs in environmental hygiene]. The involvement of official veterinarians in tasks of environmental hygiene is demonstrated under differentiation of the following working-fields: animal waste-removal, animal health protection, animal welfare, and controlling of foods of animal origin. Thereby it results that nearly all sections of public veterinary affairs are connected with environmental aspects to a high degree.


[Requirements for the education, continuing education and additional education in the area of veterinary environmental hygiene]. In view of the future of our profession it is required urgently to improve the education, continue studies and special training in the field of veterinarian environmental hygiene energetically. At this the both subjects "Animal hygiene" and "Foodstuffs hygiene" combined with the epidemiology are the central nucleus. To make plain the veterinarian engagement in this field it is proposed to introduce, a lecture in ecology and to designate the subject "Animal hygiene" in future as "Environment and animal hygiene".


[Research in veterinary environmental hygiene]. The course of development of the initial subject "Veterinary Hygiene" to the reduction and specialization limited to "Animal Hygiene" and the recent extension into the broad field of environmental hygiene is described. This is done with reference to books and other publications which point the way ahead for research of veterinary medicine in environmental hygiene and thus prove the increasing importance of that subject. A survey is given over current German research in that field, future demands as well as the necessary extension of environmental hygiene in graduate and post-graduate teaching of veterinary students as well as further professional training of veterinarians.


[Changes in the dynamics of viral infectious diseases]. Modern methods of molecular biology are suitable to analyse evolutionary processes in the area of infectious diseases. Visible expression of this evolution is the emergence of "new" infectious agents, e.g., canine parvovirus, the agent of the porcine reproductive and respiratory syndrome and phocine distemper virus. In addition a gradual change of disease syndromes may be observed, e.g., classical and African swine fever and myxoma. These processes are based on genomic changes of the respective infectious agents, i.e., point mutations and recombinations. Especially RNA viruses are prone to genomic change, and the plasticity of their genomes enables them to adapt to varying selective pressures. Human activities in agriculture, global commerce and tourism may enhance and precipitate microbial evolution.


[Environmental hygienic aspects of echinococcosis]. A literature review on the current situation of echinococcosis in Central Europe is given. The only final host for Echinococcus granulosus in this region ist the dog. The infection rate of dogs with E. granulosus in Central Europe is less than 1%. According to meat inspection statistics in Germany less than 0.008% of sheep, pigs and horses carry larval stages of E. granulosus. Parasitologically confirmed is the occurrence of a cattle-dog strain, the cysts of which were found in 0.26% of slaughter cattle. It is not known whether this strain infects also man as does the sheep-dog strain. Cases of cystic echinococcosis (E. granulosus) diagnosed in Central Europe are often imported from mediterranean countries. In Baden-Wurtemberg, the only state where human cases of echinococcosis are recorded, 50-100 cases are diagnosed per year. In areas with endemic E. multilocularis infection also dogs and cats may be infected with the adult worm besides the red fox. Recent investigations have revealed that not only in classical endemic areas (Switzerland, Austria, Baden-Wurtemberg, Rhineland-Palatinate) foxes have infection rates of up to 67% but that E. multilocularis-infection is also wide-spread in Thuringia, Hesse, Northrhine-Westphalia and in the southern parts of Lower Saxony. The most northern infested area seems to be the region of Detmold (infection rate of foxes 9%). The infection rates with alveolar echinococcosis (E. multilocularis) in humans even in endemic areas are low. In Baden-Wurtemberg 140 new cases became known during the past ten years.(ABSTRACT TRUNCATED AT 250 WORDS)


[Environmental and food hygiene aspects of cestode infections of humans]. Cysticercus bovis s. inermis, the larvae phase of Taenia saginata is still widely spread. Many reasons are at the origin thereof, e.g. incomplete destruction of the eggs of Taenia saginata during the waste water purification process and differing meat inspection practises. In an area with the same environmental and animal breeding and fattening conditions a yearly average of 6.49% infestation is found in one slaughterhouse, whilst the yearly average in a neighbouring slaughterhouse is 0.93%. The masseter muscles are the main site of infestation. The meat inspection legislation contributes to the infestation with tape-worms in man because of the weakening of the official inspection procedures.


[The effect of ecotoxicological processes on animal husbandry in industrial countries]. This paper describes results of ecotoxicological investigations concerning the effects of industrial pollutants on farm animals. By means of simultaneous experiments using dusts from lignite-filters and sedimentation-filters as well as sulfur dioxide (SO2) as stress models and by using studies on farm animals in pollutant-burden regions of eastern Germany it could be shown, that pollutants can lead to disorders of health, efficiency and behaviour, like o. g. heavy metals, and to contamination of food of animal origin. Dusts, resulting from combustion of lignite and SO2 initiate non specific effects in the organism and it is only difficult to determine their part on the decrease of productivity and metabolic disorder of farm animals in pollutant regions.


[Veterinary tasks in nature and species protection]. The present paper is designed to give examples of possible fields of activities for veterinary surgeons in the areas of nature conservation and the protection of species. The committed veterinary surgeon, who is eager to extend his/her specialized knowledge obtained at the university will certainly find a sensible and satisfying occupation in the fields of nature conservation, the protection of species and the environment. Creating the optimum prerequisites for these professional opportunities is yet another reason why public funding of the Institute for Game Research and Ecology is indispensable.


[Cooperation of veterinarians in legislation in the area of environmental hygiene (Germany and the European Economic Community)]. After a brief exposition of the tasks about the protection against chemical, physical, and biological environmental risks, a demonstration will be given according to deliberations of our law system about the possibilities for veterinarians to cooperate in the legislative branches. A detailed examination on former activities in the fields of pollution and the food-chain are to serve as a model for the future legislative process in Germany with the intention to strive for a harmonization of the in parts independently developed legislative branches. The external harmonization, a long-term objective, is to be found in the form of an uniform environmental law. Veterinarians take a great interest to work in advance for this uniform law in the way they bring new perspectives by cooperating in the fields of environmental hygiene and toxicology for the protection of the environmental media, of species and the food-chain. In conclusion one finds a brief documentation about the impact of the adaptation of law and the process on the EC-level.


[The effect of bacterial interference phenomena in experimental Staphylococcus hyicus infections of gnotobiotic piglets]. The prevention of exudative epidermitis could be confirmed in experimental investigations with gnotobiotic piglets when the skin first was colonized with avirulent strains of Staphylococcus (Staph.) hyicus and subsequently exposed to virulent strains of Staph. hyicus. However, locally restricted cutaneous lesions in the area of application corresponding to exudative epidermitis were seen in five of nine piglets. Using the strain Staph. sciuri the spread of virulent Staph. hyicus could not be suppressed. Such infected two piglets developed generalized exudative epidermitis. In another experiment with four piglets it could be shown, that the relative protective mechanism correlating to bacterial interference on the one hand can be influenced by the virulence of causative organisms. On the other hand it even can be abolished when skin lesions are involved. For that reason probably the utilization of bacterial interference in prevention of exudative epidermitis under field conditions is considerably limited.


Protein interactions in the calf eye lens: interactions between beta-crystallins are repulsive whereas in gamma-crystallins they are attractive. Non-specific interactions in beta- and gamma-crystallins have been studied by solution X-ray scattering and osmotic pressure experiments. Measurements were carried out as a function of protein concentration at two ionic strengths. The effect of temperature was tested between 7 degrees C and 31 degrees C. Two types of interactions were observed. With beta-crystallin solutions, a repulsive coulombic interaction could be inferred from the decrease of the normalized X-ray scattering intensity near the origin with increasing protein concentration and from the fact that the osmotic pressure increases much more rapidly than in the ideal case. As was previously observed with alpha-crystallins, such behaviour is dependent upon ionic strength but is hardly affected by temperature. In contrast, with gamma-crystallin solutions, the normalized X-ray scattering intensity near the origin increases with increasing protein concentration and the osmotic pressure increases less rapidly than in the ideal case. Such behaviour indicates that attractive forces are predominant, although we do not yet know their molecular origin. Under our experimental conditions, the effect of temperature was striking whereas no obvious contribution of the ionic strength could be seen, perhaps owing to masking by the large temperature effect. The relevance of the different types of non-specific interactions for lens function is discussed.


Dantrolene inhibits halothane-induced membrane reorganization. A study using 31P-NMR and differential scanning calorimetry. The action of the relaxing agent dantrolene on dipalmitoylphosphatidylcholine (DPPC) model membranes in the presence and absence of the general anesthetic halothane has been investigated by DSC and 31P-NMR. Dantrolene has a weak effect on both the thermodynamic and NMR parameters of the pure model membrane. When halothane is present in the system, the relaxing agent acts to counterbalance the strong anesthetic-induced membrane perturbation. This is reflected in DSC experiments by a change of the enthalpy variation (delta H) and of the main gel-to-fluid phase transition temperature (Tc) towards the values of the pure lipid system. The amount of halothane-induced small tumbling vesicles, as detected by 31P-NMR by the superposition of an isotropic line on a lamellar-type powder spectrum, is considerably reduced upon dantrolene addition. This means that the relaxing agent "cures" the membrane de-structuring action promoted by halothane. Membranes first treated with dantrolene are also protected from the halothane perturbation. So, the relaxing agent is both "curative" and "preventative" against halothane. The optimum effect is obtained for 1 dantrolene molecule per ca 34 halothane molecules. The mechanisms of action were discussed in relation to membrane fluidity.


Reactions in lipid vesicles. Pyrene excimer formation in restricted geometries. Effect of temperature and concentration. The decay of pyrene in the presence of excimers in small unilamellar vesicles of 3-sn-phosphatidyl glycerol, dipalmitoyl and 3-sn-phosphatidylglycerol from egg yolk has been analyzed with the use of models appropriate for reactions in restricted geometries. Results are presented with emphasis on probe concentration and temperature. The reaction rate in an organized lipid phase is redefined in a simple manner which allows for a simple treatment of any reaction in such environments. The analysis allows detection of pyrene aggregation in the vesicle lipidic core.


Formation of cell protrusions by an electric field: a thermodynamic analysis. This work gives a thermodynamic analysis of outgrowth extraction from the cell body by a pulling force. The results are applied for a case when the pulling force is generated by an external high-frequency electric field. Two equilibrium conditions are analyzed: internal equilibrium of an outgrowth and equilibrium between the outgrowth and the cell body. In both cases the stability of feasible equilibrium states was studied. The work shows that the curvature of an outgrowth equilibrated with a pulling electric force depends on the squared amplitude of the electric field E0(2), on the outgrowth length l and on the transmembrane pressure differential delta P, and that at a sufficiently large transmembrane pressure differential the cylindrical form of the outgrowth loses its stability. Long outgrowths are more stable than short ones. The minimal value of critical pressure differential was estimated. The work also shows that outgrowth extraction from the cell body requires that the applied force exceeds a critical value below which no outgrowth is formed. The value of the electric field at which outgrowth formation is feasible was estimated.


Determination of the partition of the tetramisole derivative (+-)-5,6-dihydro-6-phenyl-2-n-propyl-imidazo[2,1-b]thiazole into liposomal membranes by fluorescence quenching of the membrane probe 8-(2-anthryl)-octanoic acid. Fluorescence quenching has been used to study the partition of the tetramisole derivative (+-)-5,6-dihydro-6-phenyl-2-n-propyl-imidazo[2,1-b]thiazole into liposomes, consisting of a mixture of egg L-alpha-phosphatidylcholine, egg phosphatidylethanolamine and dipalmitoylphosphatidic acid (2:1:0.06 w/w/w). The tetramisole derivative quenched the fluorescence of the intramembrane probe 8-(2-anthryl)-octanoic acid. The quenching process could be rationalized by a model for dynamic quenching in which an intermediate excited-state non-emitting complex (PQ)* between neutral quencher (Q) and excited probe (P*) is involved: [sequence: see text] where kd, k-d and ki represent the rate constants of complex formation, dissociation and deactivation, respectively. The subscripts A and L denote the aqueous and lipid phases, and the asterisk indicates the excited state. Linear Stern-Volmer plots were obtained from quenching experiments of fluorescence intensities and fluorescence life-times. The slopes of the plots were dependent on the lipid volume fraction of the liposomes. Measurement of the reciprocal of the apparent bimolecular quenching rate constant at various lipid volume fractions yielded the partition coefficient Kp and the overall quenching rate constant kq[kq = kdki/(ki+k-d)] of the tetramisole derivative. The steady-state measurements were performed at three different pH-values. Time-correlated single photon counting measurements revealed a single-exponential fluorescence decay for 8-(2-anthryl)-octanoic acid in the presence and absence of quencher. The quenching results were in accordance with the model that only the neutral form is capable of partitioning into the lipid phase. Combined average values of 318 and 6.59 x 10(8) M-1s-1 were calculated for the partition coefficient and the bimolecular quenching rate constant, respectively, from the steady-state and time-resolved quenching experiments.


Conformational transition of an alpha-helix studied by molecular dynamics. Molecular dynamics simulations were performed on a 20-residue polyalanine helix and a spontaneous transition from a kinked to a straight conformation was observed. The kinetics of the transition was analyzed within the framework of the Kramers model for chemical reactions and within a random walk model. The Kramers model which is based on diffusion along a one-dimensional reaction pathway and the crossing of an energy barrier was found to be inadequate. Instead, a random walk model based on diffusion in the high-dimensional phase space of the system was found to be compatible with the data. The high dimensionality of the phase space permits the system to circumvent high energy barriers and diffuse rapidly at about constant energy, but decelerates the reaction since in the labyrinth of pathways the transition state is reached rarely.


Scanning calorimetric studies of the stability of tobacco mosaic virus and aggregates of its coat protein. The thermal denaturation of the common strain of a rod-shaped plant virus, tobacco mosaic virus, has been investigated by differential scanning calorimetry, and compared to that of various aggregation states of its coat protein and to that of three other TMV strains. The state of the virions was monitored by electron microscopy and analytical ultracentrifugation. The observed endotherms could be analysed in terms of a step-wise dissociation of the virions. The transition temperatures of the three successive structural changes increased with decreasing pH, from pH = 8.0 to pH = 5.0, although the corresponding enthalpy changes did not vary appreciably with pH. TMV-HR showed a stronger pH dependence of the transition temperatures than the other strains, probably reflecting the importance of the changes in affecting the charged amino acids of its coat protein. The first step of the dissociation, which correlates with the breaking up of the virions into three or four shorter rods, implies a conformational change of the particle that may be related to the first step of the in situ decapsidation of TMV.


Renal and adrenal responsiveness to angiotensin II: influence of beta adrenergic blockade. The increase in aldosterone secretion that occurs in response to Angiotensin II (AII) is enhanced when normal humans are in external balance on a low salt diet. The responsible mechanism has not been identified. Angiotensin converting enzyme inhibition reduces blood levels of AII and aldosterone, but does not decrease PRA or AI and does not modify adrenal responsiveness to AII in the sodium-depleted state. This study was designed to assess the possibility that the enhanced adrenal response reflects plasma renin activity (PRA), plasma AI concentration, or catecholamines acting via a beta adrenergic receptor. Nine healthy males were studied when in balance on a high sodium intake (200 mmol Na/day), a low sodium diet (10 mmol Na) and after 4 days of beta adrenergic blockade with either nadolol or propranolol. The adequacy of beta adrenergic blockade was assessed with a postural stimulus and significant blockade was achieved, somewhat more with nadolol (40 mg/day) than with propranolol (Inderal LA, 80 mg every 12 hrs). Beta blockade enhanced the renal vascular and pressor response to AII but did not modify the adrenal response to posture or to AII. This study confirms the role for AII levels in the modulation of renal vascular and pressor responses to AII and rules out a role for PRA, AI, or catecholamines acting via a beta adrenergic receptor in the modulation of adrenal responsiveness to AII.


Stimulation of Candida albicans transition by human chorionic gonadotrophin and a bacterial protein. Candida albicans, a dimorphic fungus, is involved commonly in human infections with the mycelium form more associated with pathogenicity. The influence of various hormones and a bacterial protein on the transition from blastospore to mycelium was assessed. Human luteinizing hormone (hLH), chorionic gonadotrophin (hCG), and an hCG-like material purified from a bacteria, Xanthomonas maltophilia (PCG), were able to increase the rate of transition when compared with the controls. The effect of the two hormones and the bacterial peptide were specific, as human follicle stimulating hormone (hFSH), thyroid stimulating hormone (hTSH), growth hormone (hGH), prolactin (hPrl) and rat and bovine LH (rLH, bLH), and bovine albumin and gamma globulin did not affect the transition. The binding of 125I-hCG or 125I-LH to spheroplasts of Candida albicans were competitively displaced by hCG, hLH, and PCG. Scatchard analysis of binding of all three ligands revealed two binding sites with a high-affinity nM Kd. Thus, hCG, hLH, and PCG induce transition of Candida albicans from a blastospore state to a mycelium form, suggesting that these hormones may modify the pathogenicity of Candida albicans.


A brief survey of pineal gland-immune system interrelationships. The present paper summarizes evidence that support the hypothesis of the existence of bilateral interactions between pineal gland and the immune system. Both in vivo and in vitro experiments show that the pineal gland, via its hormone melatonin, enhances immune function. Mechanisms involved in this immunostimulatory effect are not well understood, but some evidence suggests the existence of specific binding sites for melatonin on immune cells. Moreover, the release of opioid peptides and interleukin-2 by T-helper cells may also participate in this mechanism by activating, at least natural killer activity and antibody-dependent cellular cytotoxicity. Some immune signals, i.e., gamma-interferon, may be involved in regulating pineal function, thereby representing a regulatory mechanism in the opposite direction. The physiological and clinical significance of these data remains to be studied.