Switching of a mating-type a mutant allele in budding yeast Saccharomyces cerevisiae.

Aimed at investigating the recovery of a specific mutant allele of the mating type locus (MAT) by switching a defective MAT allele, these experiments provide information bearing on several models proposed for MAT interconversion in bakers yeast, Saccharomyces cerevisiae. Hybrids between heterothallic (ho) cells carrying a mutant MAT a allele, designated mata-2, and MAT alpha ho strains show a high capacity for mating with MATa strains. The MAT alpha/mata-2 diploids do not sporulate. However, zygotic clones obtained by mating MAT alpha homothallic (HO) cells with mata-2 ho cells are unable to mate and can sporulate. Tetrad analysis of such clones revealed two diploid (MAT alpha/MATa):two haploid segregants. Therefore, MAT switches occur in MAT alpha/mata-2 HO/ho cells to produce MAT alpha/Mata cells capable of sporulation. In heterothallic strains, the mata-2 allele can be switched to a functional MAT alpha and subsequently to a functional MATa. Among 32 MAT alpha to MATa switches tested, where the MAT alpha was previously derived from the mata-2 mutant, only one mata-2 like isolate was observed. However, the recovered allele, unlike the parental allele, complements the matalpha ste1-5 mutant, suggesting that these alleles are not identical and that the recovered allele presumably arose as a mutation of the Mat alpha locus. No mata-2 was recovered by HO-mediated switching of MAT alpha (previously obtained from mata-2 by HO) in 217 switches analyzed. We conclude that in homothallic and heterothallic strains, the mata-2 allele can be readily switched to a functional MAT alpha and subsequently to a functional MATa locus. Overall, the results are in accord with the cassette model (HICKS, STRATHERN and HERSKOWITZ )977b) proposed to explain MAT interconversions.


Gene conversion of the mating-type locus in Saccharomyces cerevisiae.

Tetrad analysis of MATa/MAT alpha diploids of Saccharomyces cerevisiae generally yields 2 MATa:2MAT alpha meiotic products. About 1 to 1.8% of the tetrads yield aberrant segregations for this marker. Described here are experiments that determine whether the aberrant meiotic segregations at the mating-type locus are ascribable to gene conversions or to MAT switches, that is, to mating-type interconversions. Diploid strains incapable of switching MATa to MAT alpha, or the converse, nevertheless display changes of MATa to MAT alpha, or the reverse. These events must be attributed to gene conversion. Further, we suggest that MATa and MAT alpha alleles may represent nonhomologous sequences of DNA since they fail to display postmeiotic segregations.


Mapping chromosomal genes of Saccharomyces cerevisiae using an improved genetic mapping method.

A triploid (3n) strain of Saccharomyces cerevisiae was constructed carrying a standard marker on each of chromosomes 1 through XVII in the -/+/+ configuration. This is called a "supertriploid." Meiotic spores from this strain (n + approximately n/2) were mated with a haploid (n) carrying an unmapped mutation. Meiotic analysis of each zygote clone (2n + approximately n/2) produced in this way resulted in elimination of an average of 4.2 chromosomes as the possible location of the unmapped marker. The distribution of extra chromosomes in the 2n + approximately n/2) strains was nearly random. Meiotic segregrants of these crosses carrying the unmapped mutation in the -/+ configuration were then crossed with multiply marked haploid strains to further narrow the possible location of the unmapped mutation to a single chromosome. Scoring of markers by complemention tests was simplified by mating spore clones with mixtures of a and alpha strains, each pair carrying the same set of markers. Using this new, more rapid method ("supertriploid mapping"), eight genes required for the maintenance of the killer plasmid were located on the genetic map of S. cerevisiae.


Physiological characterization of polar Tn5-induced isoleucine-valine auxotrophs in Escherichia coli K.12: evidence for an internal promoter in the ilvOGEDA operon.

The properties of 22 isoleucine-valine auxotrophs induced in Escherichia coli K-12 by the transposable element, Tn5, were characterized on the basis of growth requirements, cross-feeding behavior, and enzyme activity. Mutants defective in ilvA, ilvC, ilvD and ilvE were found. Mutation in ilvE were not completely polar on ilvD and ilvA enzyme activities (that is, ilvE mutants possessed a low constitutive level of expression of the enzymes coded by ilvD and ilvA), while mutations in ilvD were completely polar on ilvA enzyme activity. The data suggest that there is an internal promoter between the sites of Tn5 insertion in ilvE and ilvD.


Deletions generated by the transposon Tn10 in the srl recA region of the Escherichia coli K-12 chromosome.

A negative regulatory gene for the srl operon (srlR) was recognized by the characteristics of an insertion mutation generated by the transposon Tn10 determining tetracycline resistance. This finding is discussed in light of previous hypotheses on the regulation of the srl genes, which mediate metabolism of glucitol (i.e., sorbitol). Mapping showed that the order of genes in this region is: srlR srlD srlC recA alaS. Using two different methods, five mutations of both srl and recA were detected. The phenotype conferred by these mutations, UV sensitivity and extreme recombination deficiency, is characteristic of standard recA point mutants. Three of the mutations were deletions that also removed the genes for tetracycline resistance of the nearby transposon. A fourth mutation ended at a distance from Tn10 sufficient to allow separation of the two by recombination following P1 transduction; our tests did not allow us to conclude whether this mutation was an inversion or a deletion. The fifth mutation was a deletion that seemed to end immediately adjacent to the boundary of Tn10, proximal to recA. Mechanisms for the generation of these srl recA mutations are discussed.


Suppression of the formation of polygenotypic recombinant colonies by a maf mutation in mating with HfrH.

W3011, a Cavalli-type Hfr (HfrC), was mated with F-KY9474, maf-1, which cannot maintain F or F-like plasmids, and with F-OU9474, Maf+, a spontaneous revertant of KY9474. The recombinant colonies obtained were 100% monogenotypic from KY9474 and 90% monogenotypic from OU9474. On the other hand, in matings with OU11, a Hayes-type Hfr (HfrH), and these two F- strains, recombinant colonies derived from KY9474 showed only 22% polygenotypic recombinant colonies; whereas, those derived from OU9474 showed a high production rate (57%) of polygenotypic recombinant colonies. Among the polygenotypic recombinant colonies derived from KY9474 maf-1, 50% contained three or more recombinant types. These were probably derived from a small fraction of Maf+ revertants in the KY9474 population, as suggested by the results of mating this strain with M80, an F' strain that contains an amber mutation in traH. These results support the hypothesis that the donor DNA fragments derived from an HfrH can undergo a limited replication in the recipient to produce polygenotypic recombinant colonies, whereas those derived from HfrC cannot.


Genetic analysis of gamma-ray mutagenesis in yeast. I. Reversion in radiation-sensitive strains.

The frequency of revertants induced by 60Co gamma rays of the ochre allele, cyc1-9, has been measured in radiation-sensitive strains carrying one of 19 nonallelic mutations and in wild-type strains. The results indicate that ionizing radiation mutagenesis depends on the activity of the RAD6 group of genes and that the gene functions employed are very similar, but probably not identical, to those that mediate UV mutagenesis. Repair activities dependent on the functions of the RAD50 through RAD57 loci, the major pathway for the repair of damage caused by ionizing radiation, do not appear to play any part in mutagenesis. A comparison between the gamma-ray data and those obtained previously with UV (LAWRENCE and CHRISTENSEN 1976) and chemical mutagens (PRAKASH 1976) suggests that the RAD6 "mutagenic pathway" is in fact composed of a set of processes, some of which are concerned with error-prone, and some with error-free, recovery activities.


Rho and ribosome mutation interaction: lethality of rho-15 in rpsL or rpsE strains, and rho-15 methionine auxotrophy in rps+ strains of Escherichia coli.

The phenotype of Escherichia coli K-12 carrying rho-15 in the genetic background DW319 ilv lacZ::IS1 is described. Seventy-eight percent (70/90) of Ilv+ transductants acquired the following phenotype: temperature-sensitive growth on minimal salts medium, Ts+ growth on complex medium and suppression of the lac polar mutation. At 42 degrees on minimal medium, the rho-15 transductants were cross-fed by a substance diffusing from Rho+ transductants or controls. The requirement for this substance was satisfied by methionine or cystathionine, but not by any other single amino acid or combination of amino acids, by spermidine, or by mono- or divalent cationic salts.--Transduction of rho-15 into four other Ilv- recipients revealed two phenotypic patterns. Recipients with rpsL or rpsE ribosomes yielded rho-15 transductants that were Ts on all media, or Ts on minimal medium whether or not methionine was present. The effect of the ribosome on expression of rho-15 was confirmed by transduction of appropriate rps alleles into DW319, followed by co-transduction of rho-15 with Ilv+. The growth rate of double rho-15 rpsL or rho-15 rpsE strains was severely reduced at 42 degrees in comparison with strains carrying any of these single mutations. Models for rho and ribosome interaction are presented.


Genetic analysis of gamma-ray mutagenesis in yeast. II. Allele-specific control of mutagenesis.

We find that partially different sets of gene functions are required for the production of different kinds of mutations induced by 60Co gamma rays in Saccharomyces cerevisiae. This observation is very similar to others made previously with respect to UV mutagenesis (LAWRENCE and CHRISTENSEN 1978a,b, 1979) and confirms the conclusion that such distinctive patterns of genetic control reflect properties of the test alleles and their genetic locations, rather than the kinds of lesions required to revert them. The data also support the model of mutagenic repair outlined in the first paper of this series (McKee and LAWRENCE 1979), in which partially different sets of gene functions are required for the production of different kinds of mutations, the formation of mutations at different genetic sites and the induction of mutations by different mutagens.


Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene.

We have constructed a plasmid, YEp13, which when used in conjunction with transformation in yeast is a suitable vector for isolating specific yeast genes. The plasmid consists of pBR322, the LEU2 gene of yeast, and a DNA fragment containing a yeast origin of replication from 2 mu circule. We have demonstrated the utility of this cloning system by isolating the yeast gene encoding the arginine permease, CAN1, from a pool of random yeast DNA fragments inserted into YEp13.


Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments.

A system of biological containment for recombinant DNA experiments in Saccharomyces cerevisiae (Brewer's/Baker's yeast) is described. The principle of containment is sterility: the haploid host strains all contain a mating-type-non-specific sterile mutation. The hosts also contain four auxotrophic mutations suitable for selection for the various kinds of vectors used. All vectors are derivatives of pBR322 which can be selected and maintained in both yeast and Escherichia coli. The system has recently been certified at the HV2 level by the National Institutes of Health.


Active and inactive renin in pregnancy and in women on estrogen-containing oral contraceptives.

Total and active renin concentrations (TRC and ARC) were determined in pregnant women and in women on estrogen-containing oral contraceptives to study the variation of plasma renin forms in pregnancy. TRC was already elevated in the first trimester. After that TRC increased consistently reaching the maximum in the third trimester. The ratio of inactive renin concentrations (IRC) to TRC was between 20 and 30% throughout gestation. Therefore, it was supposed that the development of the placenta or the enlargement of the uterus do not affect the ratio of IRC to TRC too much. In women on oral contraceptives in whom plasma renin activity was increased due to elevation of renin substrate, the ratio of IRC to TRC was almost the same as that in normal controls. From these results, it was suggested that the development of the placenta and the enlargement of the uterus do not play an important role in the variation of plasma renin forms, although remarkable changes are observed in the renin substrate and total amounts of renin in pregnancy.


[Complications caused by the plastic material used in a 2-stage flexor tendon grafting operation].

After the use of the silicone-rod method for the two-stage flexor tendon grafting without complications in more than 120 cases, there were found -- although an identical technique was used -- some cases of inflammatory reaction compelling the earlier removal of the rods and leading to poorer results. Tests showed that there are silicone-rubber rods on the market which differ widely in their physical qualities and, therefore, also show differences in their compatibility with tissue.


Calculation of disease susceptibility gene frequency in insulin-dependent diabetes mellitus.

An analysis of HLA-linked genetical factors conferring susceptibility to IDDM is reported. On the basis of population and family studies a recessive mode of inheritance of disease susceptibility provided by an assumption of HLA-B8-linked DS gene was observed. The characteristic component of the immunogenetical background was the high frequency of HLA-B8 (0.208) and the HLA-A1, B8 haplotype (0.134) (linkage disequilibrium D = 0.1031), reminiscent of that found also in other disorders with autoimmune features, such as Graves disease, SLE, etc. Considering the HLA-B8 and IDDM association, the DS gene frequency (pD = 0.25) was estimated and the gametic association between HLA-B8 andu DS gene was calculated. The low value of penetrancy (4.8%) revealed the important role of non-HLA-linked genetical and environmental factors. The HLA-linked genetic factors in question might be responsible for an inclination to several kinds of autoimmune disorders.


Role of the kidney in the inactivation rate constant of urokinase activity.

The role of the kidney in the inactivation rate constant of UK activity (ke) was examined experimentally. The results obtained were as follows. There was a remarkable divergence in FA between the abdominal aorta and renal vein. When the bilateral renal arteries and veins were ligated, ke was 0.38, in contrast to 0.79 in the normal state. When UK was infused into rabbits with ligated renal vessels, the FA lasted for an increased period after the termination of UK infusion. These findings suggest that the kidney functions as an inactivator of UK.


[Pharmacological studies of 4-ethoxy-2-methyl-5-morpholino-3(2H)-pyridazinone (M73101). (2). Anti-inflammatory activity (author's transl)].

The anti-inflammatory activity and the mode of action of M73101, a new non-steroid analgesic anti-inflammatory agent, were investigated in experimental animals and compared with those of reference drugs. M73101 inhibited the increase in vascular permeability induced by acetic acid and its activity was more potent than that of phenylbutazone. M73101 showed a marked inhibitory effect against rat paw edema induced by various phlogistic agents (carrageenin, dextran, histamine, serotonin and bradykinin) and the activities were equal to or more potent than those of aminopyrine, mepirizole and tiaramide HCl. M73101 also inhibited the edema induced by mustard, scalding and anti-rat rabbit serum in rats. In addition, the anti-edematous effect of M73101 on carrageenin-induced rat paw edema was not influenced by spinalectomy or adrenalectomy, indicating that the anti-inflammatory action of M73101 was not mediated by the central nervous system and the adrenals. Local and oral administration of M73101 inhibited significantly the leucocyte migration into the fluid of CMC pouch in rats and the activity was more potent than phenylbutazone, suggesting that the anti-inflammatory effect of M73101 was due to the direct action at the inflamed site. On the other hand, M73101 did not show any marked activities on the experimental chronic inflammatory models. From these results, it is suggested that M73101 may be useful for clinical application as a basic analgesic, anti-inflammatory drug with remarkable anti-inflammatory activity in acute and subacute cases. The mechanism of the anti-inflammatory action of M73101 probably involves inhibition of an increase in vascular permeability and leucocyte migration.


[Disorders of hemostasis in intrauterine fetal death. Pathogenetic and therapeutic considerations].

When a dead fetus has remained in utero, an abnormality in haemostasis can be observed depending on the duration of retention. The most significant symptom is hypofibrinogenemia. Publications on the subject show in addition to a variety of symptoms a non-uniform pathogenetic pathway of the haemostatic disorder: Both an isolated hyperfibrinolysis and disseminated intravascular coagulation (DIC) with subsequent secondary activation of fibrinolysis have to be taken into consideration of the pathomechanism of the haemostasis defect. The triggering of DIC is possibly connected to the presence of the dead fetus and placenta, a primary hyperfibrinolysis seems to be induced by the mechanical factor of labors or curettage. The management of the haemostatic failure should be directed at the pathogenetic considerations; the appropriate treatment may be life-saving, when the haemostatic conditions become precarious inducing a bleeding tendency.


[Anti-inflammatory actions of proteases, bromelain, trypsin and their mixed preparation (author's transl)].

Anti-inflammatory actions of proteases, bromelain (BR), trypsin (TR) and their mixed preparation (KT) were studied mainly in rabbits using various experimental test methods. Inhibitory action of edema formation induced by carrageenin was observed to be dose dependent with oral administrations of KT. This inhibitory action of KT was more remarkable than actions of BR and TR, suggesting a possible synergism between the latter two. Such action was also observed with non-steroidal anti-rheumatic drugs, phenylbutazone (PB), indomethacin and acetylsalicylic acid. Oral administration of KT exerted definite inhibition or a tendency toward inhibition against paw edema induced by dextran, histamine or egg albumin or skin edema induced by anti-rabbit serum and thermal stimulation. Furthermore, inhibition of vascular permeability increase induced by histamine and bradykinin as well as a tendency toward inhibition against protein exudation in CMC-pouch method were observed. On the other hand, contrary to PB, potent inhibitory action was not manifested in the persistent proliferative inflammation models, the granuloma formation induced formalin soaked filter paper and cotton pellet and the mustard edema. Therefore, it can be deduced that the inhibitory action of KT against edema formation may be dependent mainly on the inhibitory action of vascular permeability increase and the anti-inflammatory action may be specific for acute exudative inflammation.


New ideas about self-tolerance and auto-immunity.

Recent advances in our understanding of B cell ontogeny strengthen the theory that self-tolerance is generated principally by clonal deletion. B cells pass through a stage: the "baby B cell", during which they are particularly susceptible to receptor cross-linking. Further evidence of clonal deletion comes from studies on the maintenance of the liver differentiation alloantigen F. In spite of this accumulating evidence room can still be found for the concept of immunological silence: it is a prediction of the dual recognition theory of T cell receptors that differentiation macromolecules which occur only on the surface of Ia negative cells cannot be seen by T helper cells.


The biological role of the complement system and the clinical importance of complement measurements.

Properties of serum proteins belonging to the complement system, two pathways of the complement activation (classical and alternative pathway) as well as the physiological role of the complement system are discussed. Complement has essential importance in some physiological processes: In the induction of the humoral immune response, in the elimination of immune complexes and in the protection against bacterial and viral infections. After a short discussion of the genetics of the complement system, the principle and possibilities of clinical applications of the complement measurements are described. Finally, different approaches to the therapeutic manipulation of the complement system are discussed.


Natural killer and tumor recognizing lymphocyte activity in tumor patients.

Several mechanisms can lead to the killing of a target cell by lymphocytes. In order to study immunologically specific phenomena it has to be ensured that the natural killer (NK) effect does not operate in the system. Using targets which are sensitive to NK, the effector populations have to be depleted of lymphocytes with such potential. The blood lymphocytes of tumor carrying patients often have reduced NK activity. In two assay systems anti-tumor autoimmune reactivity has been demonstrated (the majority of tested patients had lung carcinomas or osteosarcomas). The tests were: 1. Induction of blastogenesis in blood lymphocytes by in vitro confrontation with autologous biopsy cells. 2. Lymphocyte mediated killing of autologous biopsy cells in short term in vitro assay. Cross reactivity between patients was rare which indicates either that the putative tumor antigens are individual specific or their recognition is restricted by histocompatibility.


Metabolic and behavioral differences between dieting and intestinal bypass.

The present studies were designed to compare physiological and behavioral changes produced by weight loss induced with dieting and the weight loss which follows intestinal bypass. A group of 7 grossly obese individuals were hospitalized in a metabolic unit and studied at their initial weight after 4 weeks on a hypocaloric diet and again following a comparable weight loss after intestinal bypass surgery. The score indicating depression increased after dieting, but returned to initial levels after bypass. A number of other behavioral changes were recorded including a reduction in the time spent thinking about food, the time when the individual felt hungry and a greater percentage of time when they felt "full". After bypass, the patients also selected and ate smaller quantities of food. There were no metabolic differences following the period of dieting. Among the metabolic changes after bypass were an increase in glycerol and a decrease in insulin. The possible relationships between the metabolic and behavioral changes have been reviewed.


Effect of somatostatin on bile-induced acute hemorrhagic pancreatitis in the dog.

In 21 female Beagle dogs an experimental pancreatitis was induced by injection of bile into the pancreatic duct system. Beside controls, dogs received 62.5 micrograms/h cyclic somatostatin (SRIF) a continuous i.v. infusion starting with a bolus of 250 micrograms 15 minutes before or 2 hours after bile injection. Following blood parameters were determined: lipase, amylase, blood count, minerals, glucose, insulin, gastrin, secretin and CCK. Two controls died within 24 hours, the others were sacrificed after 48 hours. All pancreata were examined morephologically. The controls developed all clinical signs of acute hemorrhagic pancreatitis, whereas all SRIF-treated dogs were in much better general condition. Lipase and amylase increased in all groups. In the controls insulin, gastrin and secretin remained unchanged and CCK rose slightly. SRIF-treatment diminished insulin, CCK and the test meal-induced increase of secretin. At autopsy the pancreata of the controls were nearly entirely apoplectic. The SRIF-treated dogs showed less damage of the pancreas and no severe hemorrhagic necrosis was noted. The beneficial effect of SRIF cannot only be due to an interaction with intestinal hormones. An additional direct protective effect on the exocrine parenchyma is proposed to exist.


Growth hormone reserve capacity in Turner's syndrome.

In 11 untreated and 6 oestrogen-treated Turner's syndrome patients, the changes in the serum growth hormone level were studied following the induction of hypoglycaemia with insulin. The growth hormone was measured with a radioimmune assay technique. The growth hormone peak value measured in healthy females was 54.32 +/- 17.17, in untreated Turner's syndrome was 14.90 +/- 3.71, and in oestrogen-treated Turner patients was 33.38 +/- 9.22 microU/ml (average +/- standard error). On the basis of the results, a role is attributed to the decreased growth hormone reserve in the low growth of Tuner's syndrome patients.


Perfused liver carnitine palmitoyl-transferase activity and ketogenesis in streptozotocin treated and genetic hyperinsulinemic rats. Effect of glucagon.

Perfused liver carnitine palmitoyl transferase (CPT) activity and ketone body output were determined in streptozotocin -- treated and untreated Sprague-Dawley and Zucker rats. Streptozotocin enhanced liver ketogenic capacity and CPT activity in both these strains. No difference was observed in CPT activity or in ketone body production between the fatty and lean Zucker strains. Glucagon, added directly to the perfusate, had no influence on ketone body output and only in the livers of obese Zücker rats increased CPT activity.


Testicular function in patients with spinal cord damage.

Vaying degrees of testicular dysfunction are found in men with traumatic spinal cord damage. Eighteen paraplegic men have been studied and the gonadotropin response to luteinizing hormone-releasing hormone (LRH) measured. Basal serum testosterone estimations were made and in eight of the patients testicular testosterone reserve was assessed by the testosterone response to human chorionic gonadotropin (HCG). Testicular biopsies were performed in seven cases. In three of these patients, the testicular biopsies were abnormal. Five of the patients had elevated Follicle stimulating hormone levels and abnormalities of Luteinizing hormone kinetics were found in the same five patients. There was no significant difference between the plasma testosterone levels of the paraplegic patients when compared to the control group. In all the patients tested, there was an adequate testosterone reserve, and this included the three patients with the abnormal testicular biopsies. No relationship was found between the level of cord lesion and any of the hormonal parameters measured. This study confirms the primary nature of the seminiferous tubular damage which occurs in some patients with paraplegia.


Dynamic study of the blood viscosity of insulin-dependent diabetics by means of an artificial pancreas.

The blood viscosity of 15 insulin-dependent, poorly controlled, diabetic subjects was determined by using a microviscosimeter at low shear rates, with cylindrical cuvettes of the Couette type. It was found that the blood viscosity of these diabetics was more elevated than that of control patients (p less than 0.001). In ten diabetics, the return to a strict metabolic control over a period of more than 24 h by means of an artificial pancreas resulted in a significant systematic lowering of blood viscosity. These results suggest that the metabolic control of diabetes influences blood viscosity, and they underline the importance of an artificial pancreas for the dynamic study of the factors affecting blood viscosity during the course of diabetes.


D-Trp8-D-Cys14-somatostatin--demonstration of its differential suppressive activity in juvenile diabetics.

In 8 insulin-dependent diabetics, the effect of D-Trp8-D-Cys14-somatostatin on blood glucose, growth hormone, and glucagon levels as well as on insulin requirements from an artificial endocrine pancreas was studied during a balanced meal. The somatostatin analogue was infused at a rate of 25 microgram/h preceeded by a bolus injection of 25 microgram 30 minutes before ingestion of the meal. At this dose the analogue had no effect on glucagon levels and insulin requirements from the artificial pancreas. On the other hand, there was a significant lowering effect on fasting blood glucose levels, possibly indicating a direct inhibition of hepatic glucose production. Furthermore, there might be a slight effect on growth hormone levels, as was demonstrated by a rebound increase after termination of analogue infusion.


The artificial beta cell (Biostator) in the adjustment of instable diabetics--results after 20 months.

In 55 poorly controlled insulin-dependent diabetics, we tried to discover criteria for an improvement of metabolism by means of the "artificial beta-cell" (Biostator). To this end, during the first 24 h of hospitalization, blood glucose was monitored continuously under conventional insulin therapy (monitoring period). Insulin requirement was determined during the next 24 h by the artificial beta-cell (feedback period). Corrections of diabetes regimen were made with reference to the insulin consumption during the feedback period and to the extent of the postprandial blood sugar increases and decreases during the monitoring period. The resulting new diabetes regimen led to a significant improvement of the daily blood sugar profiles.


Insulin treatment and state of control before, during, and after connection to a glucose controlled insulin infusion system (Biostator).

Nine insulin-dependent, juvenile-onset diabetics were treated for more than twenty four hours with an artificial beta cell (Biostator). Using the amounts and profile of the insulin delivered by the machine as a guide for subsequent subcutaneous therapy, a better state of control was obtained. However, with the algorithm constants used, we found the Biostator to deliver more insulin (average 36%) than needed for subcutaneous therapy. Repeated oral glucose tolerance tests show that constants used in the algorithms might be optimized.


Use of glucose-controlled insulin infusion system for improvement of subcutaneous insulin regimen.

The use of an artificial pancreas for blood glucose monitoring and feedback correction for evaluation and improvement of subcutaneous insulin therapy facilitates the process of finding an optimal therapy regime for the individual patient. The frequency of hypoglycemic episodes can be reduced while maintaining good control, and hospital stays can be considerably shortened. This procedure is particularly useful in achieving tight control in pregnant diabetics.


The external artificial pancreas: an instrument to induce remissions in severe recent juvenile diabetes. Comparison with a preprogrammed insulin infusion system.

Remission of juvenile insulin-dependent diabetes is a rare, temporary, and partial phenomenon which seems to be related to an improvement of the residual insulin secretion supported by prompt and rigorous insulin therapy. Thus, remissions allowing the replacement of insulin by oral drugs were attempted in 23 insulin dependent ketotic juvenile diabetics (age 10 +/- 2 years) of recent onset (apparent duration of diabetes 71 +/- 5 days) treated by an external artificial pancreas during 5 +/- 1 days and compared with 10 control diabetics treated by a less effective technique (preprogrammed insulin pump without feedback control) during 6 +/- 1 days. 18 (78%) remissions of long duration (1-26 months) occurred after artificial pancreas compared with 3 (30%) in the control group. Measurement of daily urinary C-peptide excretion confirmed the improvement of the residual insulin secretion in patients with insulin-induced remissions. Thus, the excellent blood glucose control given by an artificial pancreas seems necessary to lead to much more frequent remissions of diabetes than usually reported.


Use of an artificial beta cell in surgery.

Using a Biostator glucose-controlled insulin infusion system to monitor blood glucose during surgery, we have shown that both nondiabetic and diabetic patients have a tendency towards hyperglycemia during surgery. This appears to be due to suppression of endogenous insulin secretion as measured by serum C-peptide levels. Some diabetic patients maintained relatively normal glucose values during surgery when infused with saline alone and not given glucose or insulin, but 2 of 5 were not well controlled by this means. Hyperglycemia in both diabetic and non-diabetic patients was related to the rate of infusion of exogenous glucose. The biostator glucose-controlled insulin infusion system could be used in feedback mode as an apparently safe and effective means of controlling blood glucose during surgery on diabetic patients.


Application of an artificial endocrine pancreas in the management of the diabetic surgical patient.

Application of the artificial endocrine pancreas in 12 patients undergoing total duodenopancreatectomy and 3 diabetics in whom different operations were performed proved to be safe for the patients with respect to blood glucose control and prevention of ketosis. In the postoperative period, essentially normal blood glucose values were obtained despite high caloric parenteral nutrition.


Therapeutical application of artificial beta-cell in surgery and obstetrics.

An artificial beta-cell (Biostator) was used to control blood glucose concentration in six insulin-dependent diabetics who underwent surgical procedures and in five diabetic women during both vaginal childbirth and Caesarian section. In all cases, an optimal glycometabolic control was achieved before, during, and after surgery and delivery.


Use of an artificial endocrine pancreas in diabetic pregnancy.

Insulin demand of 12 pregnant diabetics has been investigated with an artificial endocrine pancreas. A rise in insulin requirement during pregnancy which can be attributed to the effort of reaching normoglycemia and to the effect of contrainsular hormones has also been observed by this objective method. Assessment of basal insulin demand during the night might be helpful in optimizing conventional therapy by using long-acting insulins for supplementing basal insulin need. According to our results, pregnancy complicated by diabetes can be foreseen as one of the main applications of a glucose-controlled insulin infusion system.


The use of the artificial pancreas in uremic diabetic patients.

Maintenance hemodialysis and renal transplantation are increasingly used for treating diabetic patients with end-stage renal failure. The use of the artificial pancreas is able to prevent large blood glucose fluctuations in these patients with atherosclerosis, advanced retinopathy or neuropathy in which hyper- and hypoglycemia are potentially deleterious. For this purpose, we have developed and are utilizing an artificial pancreas easily utilizable without special training by the staff of a dialysis unit. This artificial pancreas uses a polarographic glucose electrode with a fast response time (45 to 90 seconds), a terminal display for operator communication, and a continuous digital and analogyl display for control of the running operation. There is also a printer to display in tabular and graphical form the values at any time during the operation. In this preliminary study, 7 patients have been studied: five under repetitive hemodialysis for four hours, 3 times a week; one treated by peritoneal dialysis for 12 hours, twice a week and one controlled during, and 48 hours after, renal transplantation. The macroscopic pancreas normalizes blood glucose under these circumstances, helps in a better understanding of blood glucose homeostasis in uremic patients under dialysis, leads to a more precise evaluation of insulin needs, may help to improve the nutritional status of the patients, and has an educational value for the patient and the medical staff.


Continuous intravenous insulin therapy with a miniaturized open-loop system.

For the continuous intravenous application of insulin, a portable open-loop system was developed consisting of a delivery unit with a miniaturized pump and an insulin reservoir which is connected with an electronic control unit. The infusion rates were either preprogrammed or patient-controlled. Blood glucose control with both systems was tested in eight juvenile-type diabetics, among them two of the brittle type. Diabetic control during a 1-2-day pre-infusion period was compared with 2-3 days of continuous insulin infusion; as judged by the mean blood glucose value (MBG), the mean amplitude of glycemic excursions (MAGE), and glucosuria, all patients were significantly better controlled by the open-loop systems than by conventional therapy with subcutaneous insulin. The use of portable open-loop systems offers a promising approach to an improvement of metabolic control in insulin-requiring diabetics.


A portable, pre-programmable insulin infusion system in diabetes mellitus.

A miniaturized portable pump has been developed in order to study the effects of pre-programmed insulin infusion patterns on the blood glucose levels in juvenile-onset, insulin-requiring diabetics. Six diabetics undergoing 7 insulin infusion periods of up to 4 days were studied. The mean blood glucose was 7.0 +/- 2.3 (SD) mmol/l and the mean amplitude of glycemic excursions was 5.0 +/- 1.4 mmol/l. Success in achieving normoglycemia will depend on choosing the correct dose of insulin for infusion.


Optimal blood sugar control in labile diabetics using a portable open-loop insulin infusion system with a flexible program.

11 labile diabetics were well controlled after 2 days of an i.v., open-loop insulin infusion program consisting of constant, empirically determined, basal infusion rates (mean: 1.1 U/h) and superimposed rectangular one-hour insulin infusions between 2 and 8 U/h during the main meals. The steering unit switches automatically back to the basal infusion rate after one hour. An almost optimal blood sugar profile was already obtained on the third day of the infusion program. We believe that such a flexible, open-loop insulin infusion program would render long-term optimal blood sugar control in "labile" diabetics possible if the technological development ever allows implantation of the infusion pumps.


Clinical application of pre-programmed insulin infusion: continuous subcutaneous insulin therapy with a portable infusion system.

Strict control of blood glucose was achieved by continuous subcutaneous insulin infusion with a specially adapted Mill Hill Infuser for 3-4 days. This led to normalisation of some of the intermediary metabolites. In a few patients, subcutaneous infusions have been carried out for periods up to 3 months with home monitoring of blood glucose, without infection or lipodystrophy. Short-term improvement of metabolic control was associated with recovery of abnormal renal glomerular permeability to albumin.


Closed-loop system in evaluation of insulin infusion rates for an open-loop system.

The daily insulin requirements as calculated with a closed-loop system (Biostator), were used for insulin infusion with a portable, miniaturized pump in 6 juvenile-onset, insulin-requiring diabetics. Three diabetics were given insulin in a manner like that of the Biostator, e.g., maximal insulin infusion about 1 hour after start of meals. The MBG was 8.7 +/- 3.9 (SD) mmol/l. Three other diabetics had insulin in a fixed profile with peaks beginning simultaneously with meals. MBG of these patients was 4.4 +/- 1.9 mmol/l. Knowing the daily insulin dosage as calculated from the Biostator, normal blood glucose levels can be achieved with a fixed profile of insulin given by a portable pump.


Comparison of a perfusion syringe (Mill Hill Infusor) and a glucose-controlled insulin infusion system in the regulation of diabetes mellitus.

In a preliminary study, three unstable, juvenile diabetics were first connected to a glucose-controlled insulin infusion system (Biostator), and subsequently to a portable infusor (Mill Hill Infuser) for continuous subcutaneous insulin application. The levels of glucose and several other metabolites as well as the insulin requirement were compared. As a tentative conclusion, continuous subcutaneous application is recommended (1) for long-term therapy when several daily doses are required and (2) after metabolic compensation with a Biostator, but before conventional therapy.


The development of an implantable electrochemical glucose sensor: response to glucose in bovine serum ultrafiltrate.

A glucose sensor based on organic oxidation reactions at a platinum electrode is being developed as the key component for an implantable artificial beta cell for diabetic patients. Sensitivity of a membrane-covered platinum electrode to changing glucose concentration in bovine serum ultrafiltrate has been demonstrated, with current response as high as 60 microamperes per 100 mg/dl change in glucose concentration. Reproducibility of measurements probably will depend on satisfactory mechanical assembly of the membrane-electrode combination. An approach to selectivity based on multiple current measurements is suggested.


Development of an electrocatalytic glucose sensor.

For the determination of glucose in body fluids, the electrocatalytic sensor, besides the enzyme sensor, is quite suitable, particularly for long-term intracorporeal operation. The measuring response is disturbed by the fluctuating concentrations of the interfering substances that are simultaneously present in body fluids. However, the disturbance can essentially be controlled, as is shown in the case of amino acids. Their marked influence on glucose oxidation is suppressed at a membrane-covered sensor electrode by the use of a potential step method.


Control algorithms for artificial beta cell.

The evolution of control algorithms for closed-loop regulation of blood glucose levels is described. Because of the rapid response time of the BIOSTATOR Glucose Analyzer, a derivative algorithm can be applied to replace the previous generation of "predictor" algorithms for the calculation of dynamic insulin infusion rates.


Development of a closed-loop artificial pancreas.

A closed-loop glucose controlled insulin infusion system was developed, consisting of elements for continuous blood glucose analysis, a computer control system, and infusion systems. Improvements include decreased size, cost reduction and better performance. The algorithm used was a piecewise linear representation of the sigmoidal curve commonly employed. The apparatus has been applied to simulation of the healthy beta cell and glucose clamp studies.


Blood glucose monitoring and computer regulation by means of an artificial endocrine pancreas.

Extracorporeal blood glucose regulation has been implemented by a closed-loop system (artificial endocrine pancreas, "aep") of our own construct. Simple, highly flexible algorithms have been developed that allow smooth repair of a deranged glucose imbalance up to a period of 125 hours.


Technology of pre-programmable insulin delivery systems: continuous subcutaneous insulin infusion.

Dual-rate continuous subcutaneous insulin infusion was performed with a specially adapted Mill Hill Infuser. The infuser delivers 2 rates, a slow rate of ca. 50 microliter/h and an eightfold higher rate, designed to cover the increased requirement during meals. The high rate is engaged by pressing a button on the side of the pump, and returns automatically to the basal level after 17 min. Insulin is delivered to the subcutaneous side by a nylon cannula.


An open-loop insulin delivery device for the control of experimental diabetes.

A new insulin delivery device has been developed and tested. It includes a reservoir, a pump, and a power pack. The reservoir holds 75 ml and is coupled to a precision peristaltic pump whose delivery can be set to any one of 128 different flow rates from 0 to 80 microliter/min (+/- 1.6% over 10 months) using the flow rate controller included in the battery power pack. The system weighs 525 g, consuming 50 mW at the maximum pumping rate, proportionately less at lower rates. Ten pumps have undergone bench tests for 30 days. One has been subjected to an extended life test of 16 months without change of tubing while seven complete systems have been used on dogs to demonstrate their capability for precise long-term (up to 16 months) intravenous insulin therapy. With this system, experimental diabetes has been controlled in 7 dogs for periods now extending beyond 16 months. This device now qualifies for-long term studies on hospitalized patients with diabetes mellitus.


Technological problems of miniaturized insulin dosing devices and some approaches to clinical trials.

With the long-term goal of developing implantable insulin dosing devices, we have since 1971 developed various types of programme-controlled units for clinical research. In the initial stage, a 24-hour, fixed-programme bed-side device has been in clinical use since 1973. The good results with even labile diabetics has supplied the basis for the further development of sensorless units. Via intermediate stages, two types of miniaturized portable devices were constructed: fixed-programme and demand-programme devices. The essential equipment components have been optimized with a view to later implantability: hermetically sealed capsule for the dosing device, refilling membrane for the insulin reservoir, bubble-free drawing off of the insulin via a wick from a collapsible silicone reservoir, the stepping motor-driven roller pump of approx. 9 cm3 volume, an energy consumption of about 50 mu W, and electronic safety and alarm circuits. Because of stability problems of the insulin, the neutral insulin U-1000 used initially was changed to acid insulin U-100 (both from Hoechst).


A portable, Preprogrammable insulin infusion system with 16 rates.

An external, portable, programmable pump has been developed for long-term therapeutic i.v. insusion of drugs. The impulse-controlled system consists of a 250 g CMOS-electronic unit and an electromechanical infusor (200 g) containing a 10 ml disposable syringe. The infusion pattern during the 24-h cycle is manually programmed in 16 steps from 24 to 384 microliter per 0.5 h. The coefficient of variation of the infusate is less than 2% per 0.5 h. An alarm circuit monitors the pump speed and the reservoir volume. The infusion system can operate continuously for 7 days with a 1 Ah, 5.4 volt battery.


Insulin responses to mixed meals: comparison of an artificial beta cell and normal beta cells.

The peripheral glucose and free insulin levels seen following a mixed meal in six insulin-dependent diabetic patients whose insulin was administered by a glucose-controlled insulin infusion system (GCIIS) were compared to those of normal subjects who received the same mixed meal or who were given separately carbohydrate, protein, or fat in amounts equivalent to those contained in the mixed meal. Patients treated with the GCIIS achieved nearly normal glucose levels immediately after the mixed meal, but this was accompanied by marked hyperinsulinemia. In the period from 120 to 240 minutes after the start of the mixed meal, the GCIIS duplicates the insulin levels produced by the normal pancreas after a glucose meal and, with appropriate algorithm constants, closely approximates those seen after a mixed meal.


Influence of insulin infusion kinetics of an artificial beta cell on blood glucose control in insulin-dependent diabetics.

The influence of different control modes for insulin infusion with an artificial beta cell was examined in 41 insulin-dependent diabetics. In 21 Patients, oral glucose tolerance tests were performed with control modes characterized either by low dynamic and high static gain (type I, 10 patients) or high dynamic and low static gain (type III, 11 patients). The change from type I to type III control mode effected an increase of initial insulin infusion rates (91 +/- 59 to 313 +/- 81 mU/min 10-20 min after glucose ingestion) and a decrease of infusion rates during the following phase of the 3-hour observation period (28.2 +/- 4.2 to 18.1 +/- 2.8 U) in patients whose blood glucose curves were completely normalized. Suppression of plasma glucagon levels, observed in 5 healthy control subjects, was not fully restored to normal in these patients. In another 20 insulin-dependent diabetics, daily insulin requirements form the artificial beta cell were determined by employing two control modes (types II and III) comparable in static control but different in dynamic control. Gain of dynamic control, especially in the range of falling glucose levels, was higher in type III control mode (15 patients) than in type II mode (5 patients). These insulin requirements were compared to the insulin doses necessary for subcutaneous treatment. While intravenous insulin requirements were much higher when type II control mode was employed (78.2 +/- 10.2%), during application of type III mode, intravenous insulin requirements were only 10.8 +/- 5.5% higher than subcutaneous doses. We conclude from these data that early increases in insulin infusion rates followed by a rapid decrease seem to reduce insulin requirements after glucose ingestion. A high-gain dynamic control is the basis for this insulin infusion profile.


Variation induced by a three-days application of the artificial beta-cell on glucagon, growth hormone and catecholamine secretion, glycosylated hemoglobin, and erythrocyte 2,3-diphosphoglycerate concentration.

Five insulin-dependent diabetics with poor metabolic control were examined at the beginning and after a three-day application of artificial endocrine pancreas (AEP). Pancreatic alpha cell function evaluated by arginine infusion (0.5 g/Kg b.w. over 30 minutes) showed no significant differences between the beginning and during artificial beta cell aplication, but the increment in plasma glucagon level over basal values observed in both tests appeared significantly higher at 30 and 60 min in comparison with a control group. Growth hormone response to arginine infusion was clearly reduced in the second test. C-peptide concentration appeared very low in basal conditions and during arginine infusion; no improvement was observed after three days of AEP application. Urinary excretion of norepinephrine markedly increased at the beginning of the study, reversed almost to normal during AEP treatment, while minor changes were observed in urinary excretion of epinephrine. The Concentration of glycosylated hemoglobin, markedly higher than normally before the connection with AEP, showed a slight but significant decrease during glucose-controlled insulin infusion. Finally, 2,3-diphosphoglycerate was normal and no modifications were observed in the course of the study.


Metabolic studies during normoglycaemic clamping of insulin-dependent diabetics using a glucose-controlled insulin infusion system.

Metabolic rhythms have been studied in six insulin-dependent diabetics during subcutaneous insulin therapy, and during control of blood glucose concentration by a glucose-controlled insulin infusion system (GCIIS). In none of the subjects was blood glucose concentration consistently within the normal range during subcutaneous insulin therapy. In contrast, blood glucose concentration was within the normal range after 3.5 h of insulin delivery by the glucose-controlled insulin infusion system and remained in the normal range for the following 8 h through lunch and dinner. Mean blood glucose concentration during this time ranged from 5.31 to 7.90 mM. Following normalisation of blood glucose concentration, blood lactate and pyruvate were similar with both the GCIIS and subcutaneous insulin therapy. Post-prandial lactate peaks were delayed with the GCIIS. Alanine levels were consistently higher during control with the GCIIS compared with subcutaneous therapy, while blood ketone body and plasma NEFA levels were lower, and the premeal peaks in the lipid metabolites were delayed. It is not possible to conclude that attainment of normoglycaemia with the present generation of glucose-controlled insulin infusion systems in insulin-dependent diabetics is accompanied by total normalisation of intermediary metabolism.


The effect of three days of blood glucose normalization by means of an "artificial endocrine pancreas" on the concentrations of growth hormone, glucagon, and cortisol in juvenile diabetics.

Glucagon, growth hormone, and cortisol secretion was studied in seven male insulin-dependent diabetics under conventional subcutaneous insulin therapy and after three days of blood glucose normalization attained by the artificial endocrine pancreas (Biostator-GCIIS). The diurnal hormonal profiles under the two types of therapy were compared. Six healthy male students served as control group. A three-day period of blood glucose normalization in insulin-dependent diabetic can restore glucagon secretion to normal. Growth hormone secretion is decreased but not completely normalised. Cortisol secretion is slightly decreased. It is concluded that prolonged normoglycemia achieved by means of an artificial endocrine pancreas may completely control endocrine abnormalities in insulin-dependent diabetics.


Feedback inhibition of insulin in healthy and overweight subjects studied by use of an artificial endocrine pancreas.

Insulin self-inhibition is still a controversial subject. The majority of data is in favour of an inhibition; however, whether this mechanism is of physiologic relevance in the regulation of insulin secretion is open to discussion. We examined the effect of exogenous insulin on beta cell secretion in 16 volunteers, including 3 who were overweight. Blood glucose (BG) was clamped by means of the dextrose infusion unit of the artificial beta cell, and the secretion of the pancreatic beta cell was monitored by immunomeasureable C-peptide (IMCP) before, during, and after infusion of insulin. The subjects were divided into 3 experimental groups. The inhibition of the basal insulin secretion was examined in group I by clamping BG at the fasting level. The inhibition of the glucose-stimulated insulin secretion was examined in group II by clamping BG at a raised level. The stimulation of insulin secretion by glucose during insulin infusion was examined in group III by stepwise BG rises from the fasting level. An inhibition of the basal insulin secretion was observed in all volunteers examined according to the protocol for group I (n = 9, including 3 overweight volunteers). The lowest insulin infusion rate applied was 1.75 U/h. An inhibition occurred at this low infusion rate corresponding to 44 mu U immunomeasurable insulin (IMI) per ml of serum. However the inhibition was impaired in the overweight participants, who, in addition, were the only ones showing a rebound rise of IMCP after stopping the insulin infusion. An inhibiting effect of exogenous insulin appeared as likely in only 1 of 5 participants examined according to the protocol for group II. Sudden rises of BG abolished the inhibition, while a total or partial inhibition was found at a constantly raised BG level in both participants examined according to protocol III. We conclude that exogenous insulin inhibits beta cell secretion, depending on the BG level, the mode of glucose stimulation, and the time relation between glucose and insulin application. Physiologically occuring IMI levels in peripheral serum were sufficient to cause an inhibition. A disturbance of the negative feedback inhibition of insulin should be discussed as a pathogenetic factor of adipositas.


Human alcohol dehydrogenase ADH2 and ADH3 polymorphisms in ethnic Chinese and Indians of West Malaysia.

Human alcohol dehydrogenase ADH2 and ADH3 were investigated in liver and stomach specimens of Chinese and Indians from West Malaysia. Eight-nine percent of the Chinese carry the atypical ADH2 type, a proportion very similar to that reported in Japanese. However, among 43 Indian specimens there was not a single case of atypical ADH2. In Indians, the gene frequency of ADH13 is 0.64 and of ADH23 0.36, similar to the frequencies in Caucasians, whereas in Chinese, the gene frequency for ADH13 and ADH23 is 0.91 and 0.09, respectively. We also report some unusual enzymatic characteristics in the course of our study.


Tapping culture--an improved method for cell suspension culture.

A new culture vessel was designed for cell suspension culture. A silicone-covered magnet bar fixed by one end to the side wall of the bottle was held horizontally a short distance from the bottom. A standard type magnetic stirrer was used. In contrast to the conventional horizontal movement of "stirring" in cultures the bar moves vertically with a "tapping" motion. This improvement resulted in less cell injury, higher rate of cell proliferation and formation of fewer bubbles than in the conventional type. Nine cell types were simultaneously cultivated in tapping, stirring and stationary culture. All cell types proliferated more luxuriously in tapping cultures then in stirring cultures. Serial cultivation of cells in tapping cultures was also successful.


Effect of cell division inhibitors on polyclonal activation can vary according to the target cell used.

The effects of inhibitors of cell division on polyclonal stimulation induced either by bacterial lipopolysaccharide (LPS) or by a synthetic adjuvant, MDP, were compared, using different target cells. Doses of colchicine that prevented 3H-thymidine incorporation also prevented the induction of antibodies against TNP and against an altered self antigen: bromelain-treated mouse red blood cells (br-MRBC). Under identical conditions, incubation with cytosine arabinoside (CA) strongly prevented the induction of anti-TNP PFC and to a lesser degree anti-SRBC PFC. However, the number of anti br-MRBC PFC was unchanged even when a dose of CA which inhibits totally the incorporation of 3H-thymidine was used. Our findings indicate that the general term "polyclonal stimulation" may concern at least two different types of cell populations and therefore we strongly stress the importance of choosing similar targets in comparative experiments.


Separation of mouse T cell subsets by a fluorescent activated cell sorter using fluorescence-labeled peanut agglutinin.

Nylon non-adherent spleen T cells obtained from concanavalin A-injected mice were labeled with fluorescein-labeled peanut agglutinin and separated into peanut agglutinin-positive (PNA+) cells and peanut agglutinin-negative (PNA-) cells by a fluorescent activated cell sorter. PNA+ cells were found to exert marked suppressive effect on primary anti-sheep red blood cells antibody response, but PNA- cells did not affect the antibody response. From these results and from the sugar-binding specificity of PNA, suppressor T cells are supposed to possess abundant galactosyl residues exposed on the cell surface, which are not masked by sialyl residues.


The application of antibody coated polyacrylamide gel (immunobead) in the NBT test for the detection of ingestion and killing functions of human neutrophils.

An improved assay for the simultaneous assessment of phagocytic uptake (via immunobeads) and metabolic integrity (via NBT dye reduction) in neutrophils is described. Normal human neutrophils exhibited ingestion and dye reduction, whereas neutrophils from newborn cord blood showed ingestion with slight reduction and neutrophils from patients with chronic granulomatous disease displayed hyper-ingestion and no subsequent dye reduction.


Outpatient treatments of obesity: a comparison of methodology and clinical results.

To provide a quantitative comparison of outpatient treatments for obesity, an analysis was made of all such research published between 1966 and 1977. All articles which presented  data for five or more healthy outpatients were included. Conclusions were: (1) there has been little improvement in the clinical effectiveness of weight reduction therapy since Stunkard & McLaren-Hume's (1959) review; (2) weight losses produced by different methods, from behavior therapy to anorectic medication, are very much the same; (3) the in-treatment results of diet therapies are the largest of those reviewed and deserve further attention; (4) behavior therapy produces the best maintenance of weight losses, although data from other types of therapy are very scanty; and (5) techniques for motivating clients to adhere to treatment recommendations are needed for improvement in the future. Recommendations for improving the methodology of weight-control research are also presented.


Dietetic treatment of obesity with low and high-carbohydrate diets: comparative studies and clinical results.

In spite of numerous studies in the literature, it is still questionable as to whether the isocaloric exchange of carbohydrate and fat, in the form of a diet, leads to different degrees of weight loss. In comparative studies, obese patients given a low-carbohydrate (4.14 MJ [1000 kcal]) formula diet (diet Ia) lost 14.0 +/- 1.4 kg and those given an iso-energetic high-carbohydrate diet (diet Ib) 9.8 +/- 0.9 kg. The degree of weight loss was significantly different. Daily weight losses were 362 g and 298 g respectively. Comparative studies of high and low-carbohydrate (7.83 MJ [1900 kcal]) formula diets (diets IIa and b) with a greater number of calories did not show any significant difference. However, there was a greater mean weight loss with the low-carbohydrate diet (351 g/day) compared with that under the high-carbohydrate diet (296 g/day). Evaluation of 117 patients treated with formula diets resulted in a weight loss of over 9 kg in 102 obese patients and over 18 kg in 52 patients. The good response to the low-carbohydrate diet was partly responsible for the successful therapy.


Chemical synthesis of [des(tetrapeptide B27--30), Tyr(NH2)26-B] and [des(pentapeptide B26--30), Phe(NH2)25-B] bovine insulins.

Two analogs of bovine insulin, [des(tetrapeptide B27--30), Tyr(NH2)26-B] and [des(pentapeptide B26--30), Phe(NH2)25-B] insulin, which differ from the parent molecule in that the C-terminal tetrapeptide and pentapeptide sequences, respectively, from the B chain have been eliminated and the newly exposed residues are amidated, have been synthesized. The [des(tetrapeptide B27--30), Tyr(NH2)26-B] insulin shows potencies of 16.8 IU/mg by the mouse convulsion assay method and 10.8 IU/mg by the radioimmunoassay method. The [des(pentapeptide B26--30), Phe(NH2)25-B] insulin possesses a potency of 10.5 IU/mg when assayed by the mouse convulsion method and 14 IU/mg by the radioimmunoassay technique. The potencies of these analogs are higher than the potencies of the respective non-amidated derivatives (Katsoyannis et al., 1973, 1974). It is speculated that the gradual decline of biological activity observed as amino acid residues are eliminated from the C-terminal region of the B chain of insulin is due to the proximity of a hydrophilic carboxyl group to the hydrophobic core of the protein molecule.


Rapid separation of proteins and their higher-molecular fragments by means of Spheron ion-exchanges.

Ion-exchange derivatives are described. of a hydrophilic rigid macroporous glycolmethacrylate gel called Spheron, suitable for rapid high-performance liquid chromatography (HPLC) of proteins and their fragments. Their flow parameters are compared with those of ion exchange derivatives of cellulose and polydextran. The conditions for work with them are described (regeneration, cycling, equilibration, column packing) as well as the construction of a simple apparatus for medium-pressure ion exchange chromatography of proteins. The efficiency of these ion exchangers for the separation of proteins is illustrated with examples of chromatography of an artificial mixture of serum albumin, chymotrypsinogen and lysozyme. Chromatography of cyanogen bromide fragments of serum albumin and the A and B chains of oxidized insulin showed that the method can be applied in chromatography on higher molecular protein fragments. A review of all proteins, including technical enzymes, which have already been chromatographed on Spheron ion exchangers is also given. The prospects of Spheron ion exchangers for HPLC of proteins and their fragments are briefly discussed.


Short in vitro half-life of thymopoietin32--36 pentapeptide in human plasma.

Thymopoietin32--36 (TP5) is a synthetic pentapeptide that has the biological activity of its parent molecule, the 49 amino acid thymic hormone thymopoietin. Tritiated thymopoietin32--36 (3 /-TP5) was prepared by reductive tritiation of dibromotyrosyl-TP5. The stability of 3 H-TP5 in human plasma was studied by analyzing samples by thin-layer chromatography at different time points and quantitating the radioactivity associated with TP5 (Arg-Lys-Asp-Val-Tyr) and its tyrosyl-containing breakdown products (Lys-Asp-Val-Tyr, Asp-Val-Tyr, Val-Tyr, Tyr). In plasma (but not in saline) the pentapeptide was rapidly degraded (apparent t1/2 approximately 30 seconds) with the corresponding appearance of radioactivity associated with the other tyrosyl-containing reference compounds. These data imply that the pentapeptide, which is active in vivo, may rapidly trigger changes in responsive cells; sustained circulating levels may not be required for activity.


Description and interpretation of morphological changes in canine kidneys after preservation with hypothermic solutions and subsequent reperfusion.

The morphological appearance of canine kidneys preserved with hypothermic solutions of three different types (Rheomacrodex, Collins 3 and Sacks-II) was studied in acute autologous reperfusion models. While at the end of preservation the renal structure still seemed intact (with the exception of the Rheomacrodex model), reperfusion was associated with the manifestation of hitherto latent structural lesions. This is confirmed by light-microscopic features (hydropic swelling and destruction of the tubular epithelium), enzyme histochemical studies (positive alkaline phosphatase reaction of the Bowman space) and topo-optical reactions (loss of anisotropy of the cytomembranes in the presence of fairly well preserved birefringence of the basement membranes).


Double-blind study of prochlorperazine, chlordiazepoxide and placebo in psychoneurotic outpatients with dominant symptoms of anxiety.

This was a double-blind study designed to compare 'Compazine', Librium and placebo in a psychoneurotic outpatient population with dominant symptoms of anxiety. The patient participants were accepted for the study when their pre-treatment symptoms of anxiety met a minimum or greater score on the Hamilton Anxiety Rating Scale. The study interval was 4 weeks. Patient psychopathology was rated by the treating physician on the Hamilton Anxiety Rating Scale, the New Physician Rating List and Global Evaluation. Patients completed the Self-Rating Symptom Scale and the Profile of Mood States. All efficacy criteria ranked the study medications in the same order. Prochloroperazine was most effective, chlordiazepoxide was also active and placebo was least effective. There are statistically significant active medication versus placebo comparisons supporting desirable drug action. Chlordiazepoxide versus placebo comparisons approached statistical significance for all efficacy criteria.


Herpes zoster and acute rejection crisis of renal homograft.

The case of a patient developing acute rejection crisis 8 months after transplantation in the prodromal stage of a herpes zoster infection is reported. The joint therapeutic measures resulted in suppression of rejection and control of the infection. Complications did not occur. The case suggests a definite association between the viral infection and acute homograft rejection. The case report is followed by a critical review of the pertinent literature.


A double-blind trial with amitriptyline and lofepramine in the treatment of endogenous depression.

A double-blind trial was undertaken to compare the antidepressant efficacy and the side effects of Lofepramine with those of Amitriptyline in the treatment of endogenous depression. The study involves 22 acutely ill endogenously depressive patients. 11 patients were treated with Lofepramine and the remaining 11 with Amitriptyline. The results demonstrate that both substances are effective in the treatment of depression. However, the therapeutical efficacy of Lofepramine is significantly greater than that of Amitriptyline. Furthermore, the tolerance of Lofepramine is better than that of Amitriptyline, and the side effects of Amitriptyline, especially in blood pressure dysregulation, are greater than those of Lofepramine.


[Biochemistry of elastin].

The recent findings on the composition and synthesis of elastin are presented. Particular attention is paid to the various specific cross-links and to their formation from lysine residues in the polypeptide chains.


[ENT considerations of head and facial pain (author's transl)].

To the otolaryngologist, there are multiple causes for head or facial pain: headaches due to ear diseases; idiopathic neuralgias; "referred otalgia" involving cranial nerves V, IX, X; temporomandibular joint dysfunction; rhinological pathologies, including post-traumatic trigeminal neuralgia; "facial sympathalgias"; the styloid process syndrome; and cervical spine problems. Less known causes of head and neck pain are stressed, and emphasis placed on their diagnosis and treatment.


Sublobar atelectasis and regional pulmonary blood flow.

We studied the effects of sublobar atelectasis on regional blood flow in anesthetized paralyzed pigs. Following the washout of nitrogen from the lung with oxygen, a sublobar airway was obstructed and a peripheral segment of lung allowed to collapse. Blood flow to the atelectatic region fell from a control of 140.0 +/- 20.5 to 16.6 +/- 2.9 ml/(min . g). Basilar regions that became atelectatic spontaneously during the course of the studies had similar decreases in blood flow. Inflation of the surrounding lung by the application of positive end-expiratory pressure failed to increase blood flow and vascular conductance within the atelectatic regions. These results indicate that mechanical and hypoxic effects on vessels perfusing sublobar atelectatic regions limit the effects of interdependence from the surrounding lung. Furthermore, with inflation of the surrounding lung, increases in pulmonary shunting of blood are small.


Lack of intracellular bubble formation in microorganisms at very high gas supersaturations.

Eucaryotic unicellular (a yeast, a cellular slime mold, and various protozoans) and two multicellular (aschelminths) microorganisms were saturated with gas at high pressures and rapidly decompressed. No effect was observed with pressures of argon up to 125 atm, nitrogen up to 175 atm, and helium up to 350 atm, showing that the induced gas supersaturations did not cause intracellular bubbles to form. With 25--50 atm higher gas pressures, the decompression usually produced killing and cell rupture, although differences in tolerances existed among the various organisms. Substantial fractions of the populations survived gas supersaturations well above the threshold values for massive spontaneous nucleation of bubbles in the water. When killing occurred, external rather than internal bubbles appeared to be the cause. Even with the 300 atm argon or nitrogen pressures, yeast cells were unaffected, apparently because of the external protection provided by their cell wall. It is concluded that the gas supersaturations required for intracellular formation of bubbles generally are at least equal to and probably higher than the bubble nucleation thresholds for water or aqueous solutions.


Fine structure of gonadotrophs and prolactin producing cells in the rat pars distalis in organ culture.

A technique is described which permits the maintenance for up to 8 days in organ culture of explanted rat pituitary glands. Electron microscope studies showed that the cultured glands (whether treated with gonadotrophin-releasing hormone (GnRH) or not) were almost identical to controls in appearance, although the stellate (follicular) cells seemingly were eliminated in vitro. Gonadotrophs and prolactin-secreting cells in male and female pituitary glands were similar in appearance both in vivo and in vitro to comparable cells described by other authors. There was some evidence for an increase in the secretory activity of these cell types (particularly gonadotrophs) when the explants were treated with GnRH. The present technique thus provides a suitable and valid 'model' with which to study the effects of GnRH and steroid hormones on the release and synthesis of FSH and LH in vitro.


Genotypic characterization and clinical evaluation of an influenza A/Texas/1/77 (H3N2)-like recombinant: RIT 4199.

The RIT 4199 (H3N2) strain was obtained by recombination of PR/8/34 (H0N1) and A/Alaska/5/77 (H3N2) isolates. Its genetic composition was determined by RNA-RNA hybridization and by identification by gel electrophoresis of the double-standard RNA formed. The RIT 4199 was inoculated intranasally to 27 seronegative volunteers at a dose of 10(7.3)EID50. The results show that it is suitable for live vaccine strain.


Induction of G1- cells at high frequency.

Experimental conditions are reported on the induction of G1- cells at high frequency by ultraviolet irradiation of temperature-sensitive cells arrested in G1 phase at the restricted temperature. Neither the wild type cells of Balb/C-3T3 nor the temperature-sensitive derivative of tsA83 grown at the permissive temperature could be 'mutated' under similar conditions. One such 'mutant' (RI8) was 'inducible' from a G1+ phenotype at 33 degrees C to G1- state at 38 degrees C within one cell generation time. The 'inducible' property of RI8 from G1+ to G1- lends support to the theory that the duration of the G1+ phenotype is determined by the level of precursors for DNA synthesis. Since the G1- variants were isolated from revertants, the frequency of 'mutagenesis' appeared to be artifically high.


Injected histone antibodies interfere with transcription of lampbrush chromosome loops in oocytes of Pleurodeles.

Antibodies to calf thymus histone H2B were purified by chromatography on DEAE-cellulose and injected into oocyte nuclei of Pleurodeles waltlii. As shown by indirect immunofluorescence these antibodies cross-reacted strongly with corresponding histones associated with lampbrush chromosomes. Shortly after injection the lateral loops of the chromosomes retracted into the chromomeres and by 3 h postinjection the 'lampbrush' appearance was completely lost and the chromosomes appeared in light-microscopic preparations as rod-like structures consisting of longitudinally coalesced chromomeres. In control oocytes injected with non-immune immunoglobulins or antibodies against a ubiquitous transcript-associated protein no morphological alterations of the lampbrush chromosomes could be observed. Electron microscopic spreads of chromosomes prepared at various times after injection of anti-H2B revealed a progressive loss of transcriptional complexes from the loop axes.  Finally, higher-order chromatin configurations, like supranucleosomal globules ('superbeads') or cable-like chromatin strands 50-60 nm thick predominated, indicating complete transcriptional inactivation of all chromosomal regions. The results indicate that H2B antibodies react specifically with histones associated with the transcribed DNA of lateral loops in their native state. The resulting antigen-antibody complexes seem to inhibit progression of the RNA polymerases along the template, thus causing the premature release of transcripts, a process analogous to the stripping effect of actinomycin D. The demonstration of histones associated with heavily transcribed regions, which are not compacted into nucleosomes but largely extended, supports the current concept that unfolding of nucleosomes to allow transcription of the DNA does not involve dissociation of histones. In contrast, amplified ribosomal RNA genes are unaffected by injected H2B antibodies. This does not necessarily indicate absence of histones from nucleolar chromatin, since we do not know whether it is accessible in vivo to antibodies or whether the histone antigenic determinants are masked by the presence of other proteins. The technique of injecting specific antibodies should be widely applicable when analysing the in vivo distribution of chromosomal components at the electron-microscopic level and when studying complex metabolic processes, like the cleavage and modification of RNA, by selective inhibition of defined enzymic steps.


Protein purification using immobilised triazine dyes.

This review attempts to identify proteins which selectively interact with immobilised triazine dyes such as Cibacron blue F3GA and Procion red HE 3B. Different support matrices are compared by examining the capacities of these dyes for proteins. Various approaches to the immobilisation of triazine dyes are considered together with the use of spacers. Some theories of the mechanism of protein retardation by immobilised dyes are discussed. A number of methods are suggested for the measurement of dye concentrations and for the modification of the binding of proteins to dye columns. The variety of elution methods is compared with a view to optimizing purifications. The scope of applications is reviewed as well as the choice of dye. Some advantages of triazine dyes over other affinity ligands are given. It is concluded that although no satisfactory mechanism for the binding of triazine dyes to proteins has yet been proposed, these dyes possess considerable potential for protein purification, particularly when applied on the large scale.


Applications of chromatography in the standardization and control of biological products.

The problems posed in the standardization of medicinal biological products and in the development of official specifications for such products, e.g., in pharmacopoeias, are outlined. Information derived from bioassay can be extended and complemented by that from physico-chemical studies. The role of chromatography, including gas--liquid, thin-layer and column methods, is assessed and illustrated with examples from the literature and from the author's studies on antibiotics and polypeptide hormones.


High-pressure liquid chromatography of steroids.

After a brief discussion of the merits and limitations of high-pressure liquid chromatography (HPLC) relative to other chromatographic methods, special problems in the application to steroids are discussed. Publications on HPLC of steroids are then discussed under the headings of individual classes, arranged generally in the order of increasing polarity.


Detection of anaerobic wound infection by analysis of pus swabs for volatile fatty acids by gas-liquid chromatography.

Swabs were able to absorb enough extractable volatile fatty acids from broth cultures of anaerobic organisms for detection and analysis by gas-liquid chromatography (GLC). Similarly, volatile fatty acids were often detected in swabs dipped into liquid pus. Fifty-three liquid pus specimens were then investigated fully to determine if GLC analysis of swab samples gave the same result as microbial culture of the specimens and GLC analysis of the liquid pus. Anaerobic bacteria failed to grow from 36 and volatile fatty acids were not extracted from swabs of 31 of these pus samples but were extracted from swabs of five. Anaerobic bacteria were isolated from 17 of the specimens, and in 15, volatile fatty acids were also detected in the swab samples; in two, volatile fatty acids were absent from both swab samples and liquid pus. In this study, results by culture and GLC analysis of swabs were similar in 87% of specimens.


Significance of urinary immunoglobulins in tests for antibody-coated bacteria.

The relationship between urinary immunoglobulin levels and coating of a panel of bacteria by these immunoglobulins has been investigated. The results indicate that elevated urinary immunoglobulin levels are a distinct hazard in interpreting tests for antibody-coated bacteria as indicating upper or lower urinary tract infections.


Serodiagnosis of Trichomonas vaginalis infection by the indirect fluorescent antibody test.

The indirect fluorescent antibody test was used to detect antibodies to Trichomonas vaginalis in 200 antenatal patients. A total of 104 (52%) gave a positive reaction with antigen prepared from cultures of trichomonas isolated from seven of the patients. Antitrichomonal antibody was detected at a 1/4 dilution in 90% of patients with proven trichomoniasis, while the highest dilution at which antibody was detected was 1/32. IgG rather than IgM appeared to be the antibody class involved. Of those patients with no demonstrable trichomonal infection, 17% gave positive reactions at 1/4 dilution, while 64% had no demonstrable antibody. One of 30 children under 11 years of age gave a positive reaction.


Use of human embryo lung fibroblasts to detect a heat labile toxin of Escherichia coli from children.

In order to detect the heat labile toxin of Escherichia coli human embryo lung fibroblast cells were seeded with whole cell lysate preparations of the organism to be tested. Positive results consisted of growth inhibition and cytopathic change which were easily seen. Heat-labile toxin was produced by strains belonging to the conventional epidemic serotypes of E. coli (EEC) and by non-EEC strains. Toxin-producing organisms were detected in 20% of healthy children examined. The method is suitable for use in the diagnostic laboratory and is easily adapted to a microtitre plate system suitable for screening purposes.


Detection of human antibodies to hepatitis B surface antigen (HBsAg) by an enzyme-immunoassay for HBsAg.

We studied the feasibility of detecting antibody to hepatitis B surface antigen (anti-HBs) by an inhibition assay using the reagents of an enzyme-immunoassay for HBsAg (Hepanostika). Several modifications of the basic assay were investigated. Sensitivity was greatest when the test sample was incubated with a predetermined amount of HBsAg before the usual procedure of HBsAg detection. The presence of anti-HBs in the test sample was shown by a reduction of the solid-phase bound enzyme label. Results were obtained with a dilution series of serum samples containing anti-HBs, the anti-HBs Reference Panel of the American Bureau of Biologics, sera of hepatitis B patients, and sera of two individuals passively immunised with anti-HBs. The enzyme-immunoassay method showed at least the same sensitivity as passive haemagglutination. It was less sensitive than a commercially available radioimmunoassay (Ausab). There are no indications that non-specific reactions occur frequently. This study also revealed that the antigenaemia of acute hepatitis-B patients can be interrupted by a transient seroconversion.


Appropriateness of milk use in international supplementary feeding programs.

Recognition of lactose intolerance, debate over the limitedness of dietary energy and protein in specific settings, and the appreciation for the uniqueness and appropriateness of human milk during the 1st yr of life have prompted reexamination of the use of milk in international feeding programs. Substantial proportions of preschool and school-age children from expected populations are lactose intolerant. However, studies in this county indicate that lactose intolerant preschool and early school-age children can tolerate amounts of milk usually consumed. Older individuals with symptomatic responses to milk also will often have similarly mild symptomatic responses to hydrolyzed lactose milk. Appropriately controlled studies in developing countries are planned or are underway to assess milk tolerance. In feeding programs designed to provide a protein supplement, milk continues to be a very attractive option. The question of how much milk protein must be given and how often it should be provided to make a significant impact on a population's well-being depends on an evaluation of the data base describing protein intakes and needs for specific populations. These types of data for most populations are inadequate.


Testosterone concentrations in plasma of dairy cows with ovarian cysts.

Testosterone in plasma was measured by radioimmunoassay in cows with ovarian cysts and in cows during the estrous cycle. Concentrations of testosterone in plasma (60.8 +/- 2.7 pg/ml; mean +/- standard error) in cows with ovarian cysts were variable but within the range in cows during the estrous cycle. Mean concentrations of testosterone in plasma increased from 46.3 pg/ml at estrus to 85.1 pg/ml on day 13 of the estrous cycle. In cows with ovarian cysts, concentrations of testosterone in plasma declined within 24 h following prostaglandin F2 alpha and tended to increase by 9 days following gonadotropin releasing hormone. The role of testosterone in estrual behavior and inhibiting resumption of ovarian cycles is likely minimal.


[Photochemistry of visual pigments (author's transl)].

Recent studies of the photochemistry of visual pigments are reported. Results obtained on rhodopsine and model compounds, by means of techniques based upon continous or pulsed excitation are compared. The mechanique of the primary photochemical step of the bleaching of rhodopsine is discussed. The process is occuring on a time scale of picosecond and a new concept of proton transfer followed by a conformation charge is opposed to the classical cis-trans photo isomerisation of the retinal moiety. The origin of the electrical response of the photoreceptor cells is still controversial.


Fixed-dose combination diuretics in congestive heart failure: an evaluation.

Thirty-two patients with congestive heart failure were studied for their clinical and biochemical responses to the administration of two combination diuretic products (hydrochlorothiazide/spironolactone and hydrochlorothiazide/triamterene) and the single entity diuretic furosemide. Data from these studies revealed the following: 1. Comparison of the patients on furosemide with those receiving the combination products showed no difference in serum potassium or 24-hour potassium excretion. 2. Significant changes in blood urea nitrogen, plasma renin activity, and urinary aldosterone excretion were noted with both fixed-combination medications but not with furosemide. 3. The combination diuretics offered no clinical benefits over the single agent furosemide. 4. Therapy is best served by the use of a single effective diuretic agent for the treatment of most patients with congestive heart failure.


Darwin sees the insane.

Charles Darwin's work on human and animal expression introduced this age-old problem into psychological study. Darwin's interest in the expression of the mentally ill led him to correspond with J. Crichton Browne, a young psychiatrist, who supplied him with detailed materials concerning the insane as well as photographs of them.


Glucose-induced paradoxical hyperkalemia in patients with suppression of the renin-aldosterone system: prevention by sodium depletion.

A paradoxical transitory elevation of serum potassium concentration after intravenous infusion of hypertonic glucose has been found in 6 renal and/or hypertensive patients with suppression of the renin-aldosterone system (RAS) while on high sodium intake. Sodium restriction induced a dramatic increase in plasma renin activity (PRA) and/or plasma aldosterone (PA) in every patient, a substantial fall in the elevated serum potassium levels in 4 out of the 6 patients and a marked increase in fractional potassium excretion. During sodium restriction the glucose-induced paradoxical transitory hyperkalemia was abolished. The study confirmed the important extrarenal influence of aldosterone in the maintenance of normal potassium level in the hyperosmolal extracellular fluid and showed that: i) high sodium intake may predispose to hazardous hyperkalemia after massive glucose loading in certain nondiabetic patients with liability to suppression of aldosterone; ii) sodium restriction abolishes the glucose-induced abnormal serum potassium response.


[Fluorescence histochemical identification of axons using the GOLGI method].

Using fluorescence histochemical and GOLGI-methods afferent fibers in the albino rat's dorsal lateral geniculate nucleus (d LGN) have been investigated. We considered the following parameters: diameters of the axons, diameters of boutons, number of the boutons by 100 micrometers axon length. From comparison of the data from histochemical material with the data of the GOLGI-material follows that the noradrenergic terminals are identical with the type-4- and 5-axons. The terminal branching and synaptic contacts of the type-4- and 5-axons are discussed in connection with the aminergic innervation of this nucleus.


Effectiveness of peroral and intranasal immunization of school children with live allantoic influenza vaccine- and comparative study.

A comparative study of two preparations of allantoic live influenza vaccine, one for intranasal and the other for peroral immunization of children of school age, was peformed under conditions of a blind epidemiological trial. Previously obtained data on the safety and high immunogenicity of the intranasal vaccine variant, prepared from extremely attenuated cold-adapted strains, were confirmed. The peroral administration of the live influenza vaccine, in use in the USSR for active immunization of the adult population, also stimulated influenza immunity without producing postvaccinal reactions. Peroral and intranasal immunization with the above variants of live allantoic influenza vaccine markedly lowered in the frequency of influenza disease during an influenza epidemic, the mean index of effectiveness being equal to a factor of 2. Evidence of prospectiveness of influenza prophylaxis among school children was obtained.


[Application of 4-META on adhesive opaque resin. (Part 2) Improvement of adhesive stability (author's transl)].

We had reported 4-META adhesive opaque resin for a metal casting etched with HC1. The bonding strength was as excellent as 260 kg/cm2 after 300 times thermal cycles between 4 degrees C and 60 degrees C but it decreased to 70 kg/cm2 when immersed in water at 37 degrees C for 30 weeks. It was required to improve the stability of bonding against water penetration into the adhesive junction before the clinical application. Preparation of the passive state on the metal casting adhesive stability between this film and the opaque resin were studied in this paper. When the metal casting was dipped in conc. HNO3 for fifteen minutes, adhesive strength was 260 kg/cm2 even after a 30 weeks immersion in water at 37 degrees C or the 10 weeks at 80 degrees C. It could be concluded that the adhesive opaque resin possesses enough adhesive strength and the stability for oral application.


p-Hydroxymercuribenzoate-induced hyperglycemia: influence of pre- and post-treatment with L-leucine, tolbutamide, D-mannoheptulose, insulin and alloxan.

The aims were to see whether p-hydroxymercuribenzoate (PMB) administration affects the serum insulin concentration in mice in vivo, whether the transitory hyperglycemia induced in fed mice by treatment with PMB is affected by L-leucine, tolbutamide, D-mannoheptulose or insulin, and whether PMB affects the B-cell toxicity of alloxan. A significant decrease in the serum insulin concentration was found 1 and 2 h following PMB injection in fed and starved mice. PMB-induced hyperglycemia was abolished by pre-treatment with L-leucine and tolbutamide, but not by pre-treatment with D-mannoheptulose, or by post-treatment with insulin. Pre-treatment of fed mice with PMB caused potentiation of the initial hyperglycemia following alloxan, but inhibited the second hyperglycemic phase. These findings indicate that PMB treatment of mice has a transient inhibitory influence upon insulin secretion, and protects against the development of alloxan diabetes.


Oestrogen receptors in cell nuclei of the hypothalamus-preoptic area-amygdala following an injection of oestradiol or the antioestrogen CI-628.

Oestrogen receptors were measured by an exchange assay in cell nuclei of the hypothalamus-preoptic area-amygdala (HPA) of ovariectomized-adrenalectomized (OVX-ADX) rats following an intravenous injection of oestradiol or an antioestrogen, CI-628. Receptors were translocated to the nucleus by both compounds. Receptors translocated by the antioestrogen were specific for oestrogens; testosterone, corticosterone and progesterone were not bound by these receptors. Several properties of antioestrogen- and oestradiol-receptor complexes were compared. The time-course in cell nuclei in vivo showed that receptors were still present in HPA nuclei 24 h after administration of CI-628 but not 24 h after oestradiol. This prolonged increase of nuclear receptors after the antioestrogen treatment was attributed to the continued presence of CI-628 and its metabolites in plasma. The maximum level of receptors produced in the HPA cell nuclei following CI-628 treatment was lower than the peak level of nuclear receptors following oestradiol. The dissociation rate in vitro of nuclear antioestrogen-receptor complexes formed in vivo was more rapid at 0 degrees C than that of nuclear oestradiol-receptor complexes. This property may be related to the lower peak level of binding after CI-628 treatment and to the inhibitory actions of antioestrogens.


Varying response to luteinizing hormone of two luteal cell types isolated from bovine corpus luteum.

Luteal cell suspensions obtained by enzymatic digestion of pregnant cow corpus luteum were found to be heterogenous and mainly made up of two types of cells of different sizes. The large cells (37 micrometers, average diameter) could be separated from the small ones (18 micrometers, average diameter) by sedimentation at unit gravity in a gradient of Ficoll-bovine serum albumin. A comparative in-vitro study of the synthesis of progesterone by the two types of cells indicated striking differences between them. The average content and the synthesis of progesterone in the absence and presence of a saturating dose of bovine LH after incubation for 2 h were 0.07, 0.12 and 6.9 pg/cell for the small cells and 0.65, 2 and 10 pg/cell for the large ones. Moreover, the sensitivity to low concentrations of LH was 100 to 1000 times higher for the small cells than for the large ones. Oestradiol-17 beta at concentrations ranging from 5 X 10(-10) to 5 X 10(-4) mol/l exerted a dose-dependent inhibition on the stimulation of LH in both cell types. These results suggest a possible involvement of both cell types in the synthesis of progesterone in vivo with a greater contribution by the small cells to stimulation induced by LH. Moreover, it appears that small cell suspensions could be a useful model system for in-vitro studies of the control of the synthesis of progesterone in cow corpus luteum.


Mechanism of the first spontaneous gonadotrophin surge and that induced by pregnant mare serum and effects of neonatal androgen in rats.

The mechanism of the first (pubertal) preovulatory gonadotrophin surge was investigated in Wistar rats by measuring (1) LH releasing factor (LH-RF) in pituitary stalk plasma and in extracts of hypothalamic and preoptic tissue, (2) LH and FSH in peripheral plasma and extracts of anterior pituitary tissue and (3) the LH and FSH response to LH-RF. The mechanism of the first surge appeared broadly to resemble that in the adult. That is, the gonadotrophin surge which occurs on the afternoon of the day before vaginal opening was found to coincide with a surge of immunoreactive LH-RF in pituitary stalk plasma and a significant increase in pituitary responsiveness to LH-RF. The magnitude of the change in pituitary responsiveness was, however, less at puberty than in the adult while the peak of the LH-RF surge was higher at puberty. The surges of LH-RF in stalk plasma and gonadotrophin in peripheral plasma corresponded relatively precisely with a fall in the preoptic and hypothalamic content of LH-RF and in the pituitary content of LH and FSH, suggesting that, in contrast with the adult, the synthesis of LH-RF and gonadotrophin at puberty cannot keep up with their release. A significant increase in stalk plasma LH-RF concentration occurring concomitantly with a surge of LH could be induced on the afternoon of Day 32 of life by administering pregnant mare serum (PMS) ON Day 30 (about 10 days before vaginal opening). This, together with other evidence, suggests that the timing of the first gonadotrophin surge depends upon the capacity of the ovary to secrete oestradiol in the form of a surge. The fact that no significant increase in the pituitary responsiveness to LH-RF occurred in PMS-treated rats could account for the fact that the height of the gonadotrophin surge in these animals is only a third that of the spontaneous surge. No significant change in peripheral plasma LH or stalk plasma LH-RF concentrations was found around the time of vaginal opening or cornification in female rats given 1.25 mg testosterone propionate on Day 4 (androgenized female rats). Studies on the effect of ovariectomy and/or adrenalectomy suggested that the ovary and adrenal are involved in the timing of vaginal opening in normal but not in androgenized female animals.


Effects of photoperiod on hypothalamic luteinizing hormone releasing hormone in the male hamster.

Male hamsters were maintained on long (14 h light : 10 h darkness; 14L : 10D) or short (6L : 18D) photoperiods. Animals on short-days had reduced levels of LH in the serum and anterior pituitary gland, decreased androgen in the circulation and regressed testes and accessory sex organs. These same hamsters had significantly raised concentrations of hypothalamic luteinizing hormone releasing hormone (LH-RH). There was no significant difference in the response to exogenous LH-RH between groups maintained on long- and short-days. Castration significantly reduced levels of LH-RH in the hypothalamus in the long-day animals but had little effect on this parameter in short-day animals which had already undergone testicular regression. The increased levels of LH-RH in the hyothalami of both intact and castrated hamsters on non-stimulatory photoperiods is interpreted as a decreased release of the neurohormone which subsequently results in a decreased release of LH.