Diet, DDT, and the toxicity of drugs and chemicals. The toxic and carcinogenic effects of many compounds depend on their activation to reactive molecules in the cytochrome P-450 system of the endoplasmic reticulum of cells. Changes in dietary input alter P-450 levels in different tissues and so alter toxicity. In this way, low protein diets protect against carbon tetrachloride poisoning. Fats, proteins, and nonnutrients such as flavones, antioxidants, and contaminants like DDT all affect P-450 levels. The activated molecules may be diverted from their target sites by inactivation processes, such as epoxide hydratase or glutathione trappin. This is also under nutritional control. Low protein diets render animals sensitive to acetaminophen by reducing glutathione levels. In the sequence of events leading from initial contact of toxin with organism to eventual cell injury or neoplasm, nutritional factors are of import at every stage. Assessments of the toxicity of chemicals that do not take into account the nutritional variable in man are likely to be incorrect.


Teratogenic effects of environmental chemicals. Despite the widespread distribution of a great many chemical substances in the environment, very few have been implicated in human teratogenicity, and most of these are drugs used at relatively high biological effect levels. Although several other types of environmental chemicals such as pesticides, solvents, and metals can be shown under laboratory conditions to have some teratogenic potential, there is little evidence that these, at present ambient concentrations and conditions of exposure, represent significant hazards to human intrauterine development. An exception to the latter generalization is methylmercury which, because of peculiarities in distribution, can reach high concentration in the human diet.


Radiation injury: some aspects of the oncogenic effects. The late effects or irradiation stem from cell killing, mutation, and malignant transformation. Cancer is the major somatic late effect of exposure to low dose levels of radiation, and estimates of risk of cancer in man after irradiation are based entirely on human experience. The data for dose-response relationships for the induction of tumors by external irradiation in man have been obtained from a single exposure or a small number of exposures delivered at high dose rates. In contrast, exposure to environmental irradiation is mainly protracted over a long period of time and is delivered at a low dose rate. As yet no allowance has been made for the effect of protraction of the exposure time in estimating the risk of cancer, although an adjustment has been made in the case of estimates of genetic risk. Incidence of tumors has been the only parameter used for risk estimates, but latent period and degree of malignancy, which are probably both dose and dose-rate dependent, influence the nature of the risk from radiation. As the knowledge about the effects of low-level radiation has been accumulated and assimilated over the last 70 years, so has the concern for reasonable standards of safety. There are still problems in the estimation of radiation risks, but at least many of the relevant questions can now be framed. The problems of estimating risks for chemical carcinogens are clearly greater, but the experience gained from radiation studies should help in the design of the necessary experiments.


Influence of environmental stress on pathogenesis of sudden cardiac death. The effects of 20th-century stress on the cardiovascular system are reviewed and correlated with experimental animal models. A classic example of such stress is drawn from a study of the aerospace workers at Cape Kennedy who were shown to be exposed to excessive occupational stress. Surprisingly, the usual risk factors did not predict a greater risk, yet the population exhibited a higher incidence of sudden cardiac death and acute myocardial infarction. Acute myocardial necrosis was much more frequently demonstrated than was acute coronary obstruction of any type. Retrospective coroner's studies revealed two types of myocardial necrosis: 1) elongated, thinned or wavy fibers and 2) anomalous contraction bands. Correlation of these clinical observations with experimental data was duplicated in canine models of myocardial infarcion and/or catecholamine-induced necrosis. Catecholamines can lead to irreversible myocardial necrosis but the underlying mechanisms appear to be complex. Extrapolation of the results from the experimental and clinical studies suggests that environmental stress can lead to myocardial necrosis.


Immune responses to environmental antigens that act on the skin: the role of lymphokines in contact dermatitis. Immune responses to enviornmental agents affecting the skin may take various clinical forms, among which contact dermatitis is the most prominent representative of delayed-type hypersensitivity. Whereas in industrialized countries a relatively restricted amount of chemical agents is responsible for the majority of contact dermatitis cases, other factors from the environment such as natural flora, seasonal or nutritional factors may also play a role. Like other immune responses, contact dermatitis is strongly influenced by genetic factors and the existence of immune response genes, in part linked to the major histocompatibility complex, has been established in experimental animals. Whereas the formation of conjugates between skin-specific proteins and contactant allergens is held by some to represent an important feature in contact dermatitis, recent experiments suggest that the direct binding of contactants to monocyte and lymphocyte membranes represents the most efficient way in inducing sensitization of the T lymphocytes primarily responsible for contact hypersensitivity. At the effector level, complete inhibition of contact dermatitis and other delayed type hypersensitivity reactions by an antiserum prepared against guinea pig lymphokines (especially migration inhibition factor) offers strong evidence that lymphokines, as products of activated lymphocytes, also play a decisive role in vivo. The properties of antibodies raised against purified lymphokine fractions are reviewed. Localized contact dermatitis reactions, as well as accompanying phenomenons such as flar-up reactions and generalized maculopapular rashes, may, however, still involve other elements than T lymphocytes and lymphokines. The participation of other secondary cell types and of local antibody formation is briefly discussed.


[Des-Asp1] angiotensin II: mediator of the renin-angiotensin system? Angiotensin II and its C-terminal heptapeptide fragment, [des-Asp1]angiotensin II, influence a variety of angiotensin receptors in a qualitatively similar manner. On the basis of potency studies, angiotensin II appears to be the important mediator of the renin-angiotensin system at the peripheral arteriolar receptors to maintain arterial blood pressure. However, both angiotensin II and the heptapeptide are approximately equally potent at receptor sites in the adrenal cortex, the renal arterioles, and the juxtaglomerular cells of the kidneys. Adrenal cortical receptor affinity appears to be greater for the heptapeptide than for angiotensin II. Analogues of the heptapeptide are better antagonists than analogues of the octapeptide in blocking the steroidogenic responses to both angiotensin II and heptapeptide. Circulating plasma levels of [des-Asp1]angiotensin II appear to be low in most species; there is strong evidence, however, that local generation of heptapeptide can occur under certain conditions. It seems likely that both peptides act at common receptor sites to mediate the response to the renin-angiotensin system but more data are needed before a definite physiologic role can be assigned to the heptapeptide.


Comparative physiology of the renin-angiotensin system. Renin or a renin-like substance is found in the kidneys of many vertebrate species. It is absent from the kidneys of cyclostomes and elasmobranchs and first appears in holosteans and the bony fishes as well as in all higher vertebrate species. Juxtaglomerular cell granules also appear first in holosteans and the bony fishes while the macula densa first appears in amphibians. In telecost fishes, the corpuscles of Stannius contain Bowie-stainable granules and a renin-like pressor substance. Among classes and, in some cases, species of vertebrates, specificity in the reaction of renin with a substrate has been demonstrated. There is also some species and class variation in the angiotensin molecule since angiotensins of fishes, amphibians, reptiles and birds have chemical characteristics different from each other and from those of ammmals. A role for renin in stimulating interrenal gland steroid biosynthesis and in influencing water and ion regulation in nonmammalian vertebrates is discussed.


Contraceptive and sterilization practices and extrauterine pregnancy: a realistic perspective. Comparative data on the incidence of ectopic pregnancy among accidental pregnancies associated with failure of a contraceptive or sterilization procedure are shown in Table 16. The practical clinical significance of the data in this review is predicated upon a number of related factors. One of the most important of these is the realistic failure rate (or success rate) of each contraceptive or sterilization method. The reported efficacy of various contraceptive methods has such a wide range that we have not attempted to calculate the likelihood that a woman may experience an ectopic pregnancy within a particular time period while using a specific method. The success or failure rate of each method is influenced by such variables as (1) the conscientiousness and ability of the patient to follow instructions, (2) the true failure rate of the method itself, (3) the experience of the clinician prescribing a method or performing a surgical procedure, and (4) other factors less clearly defined. Because of these many variables, the data shown in Table 16 were calculated on the basis of the number of reported accidental pregnancies which occurred coincidentally with, or subsequent to, a specific contraceptive or sterilization modality. (formula: see text). These data do not reflect the actual rate of occurrence of ectopic pregnancy with respect to woman-months of experience. We recognize the significant influence that age, race, gravidity, and socioeconomic factors have upon the incidence of ectopic pregnancy, but were unable to control for these factors in the data presented in this review. These data reprresent only what has occurred under specific circumstances and cannot logically be extrapolated to any specific future case or study series. It is important to emphasize the necessity of constant awareness by the medical and paraprofessional personnel of the potentially increased risk to the patient of an extrauterine pregnancy should one or another of these contraceptive or sterilization procedures fail. Complacency or a false sense of security on the part of responsible medical personnel concerning women who are "protected against conception" can quickly lead to a life-threatening situation in case of an ectopic pregnancy. Prompt diagnosis and definitive treatment of the extrauterine pregnancy is vital for the successful management of this serious complication of conception.


Treatment of functional amenorrhea-galactorrhea with 2-bromoergocryptine. The present study was undertaken to investigate not only the effectiveness of bromoergocryptine therapy in 13 women with amenorrhea-galactorrhea and hyperprolactinemia without evidence of organic pathology, but also to assess the value of pretreatment evaluation in predicting the response to therapy. Sella turcica tomography, base line serum follicle-stimulating hormone, luteinizing hormone (LH), thyroid-stimulating hormone, T4, plasma cortisol levels, and the growth hormone reserve were normal in all patients. The pretreatment administration of LH-releasing factor (LRF) (100 microng subcutaneously) resulted in either a normal or excessive release of LH. On bromoergocryptine therapy, cyclic menses were reintiated in 10 of the women, while conception occurred prior to reinitiation of menses in the remaining three women. The time required for resumption of menses or conception on therapy correlated well with the magnitude of gonadotropin response to LRF. No correlation was seen with pretreatment prolactin levels nor with the degree of suppression of prolactin during bromoergocryptine therapy. In four women the mean prolactin levels during therapy were above normal, and in one patient prolactin levels approached pretreatment values during therapy. The initiation of cyclic menses despite continued hyperprolactinemia may indicate a possible direct effect of bromoergocryptine on hypothalamic LRF secretion as a partial explanation for its therapeutic action. On discontinuation of bromoergocryptine therapy, serum prolactin levels rapidly returned to pretreatment values or higher in all of the patients studied. In contrast to previous studies in which amenorrhea recurred in all patients after discontinuation of therapy, three of our patients maintained cyclic menses despite continued hyperprolactinemia. The recurrence of hyperprolactinemia after discontinuation of bromoergocryptine would indicate a persistent autonomy of the mechanisms involved. Periodic endocrine evaluation will be necessary to substantiate the presence or absence of pituitary microadenoma in these women.


Aberrant uterine response to prostaglandin E2 as a possible etiologic factor in functional infertility. The clinical entity of functional infertility still remains unexplained and largely lacking a line of proper management. The uterine response to prostaglandin E2 (PGE2) and prostaglandin F2alpha (administered by intravenous and intrauterine routes) was evaluated during different phases of the menstrual cycle in five functionally infertile women. The results (quantitative and qualitative) were compared with those elicited among 20 fertile women. There were no apparent differences between the two groups with respect to either compound (systemic or local) when given during the proliferative or luteal phase of the cycle. However, at midcycle, about the time of ovulation (days 13 to 18), there was a salient deviation in the type of response following local instillation of PGE2; in the normal fertile group there was a definite inhibition of motility, while uteri in the functionally infertile group showed marked stimulation. The possible role of this aberrant uterine response in the etiology of functional infertility is discussed.


A study of the relationship between coitus and the luteinizing hormone surge. Twenty-seven wives participating in a study of sexual activity patterns had identifiable shifts in basal body temperature and midcycle surges of serum luteinizing hormone (LH). When sexual activity data were examined using the LH surge as a centering point, it was observed that the day before the LH surge had only an average probability of coitus and organism. These findings suggest that neither coitus nor orgasm often triggers ovulation in humans. This cannot be taken as evidence that coitus-induced ovulation never occurs.


Luteinizing hormone (LH), follicle-stimulating hormone, and testosterone responses to consecutive injections of D-leucine-6-LH-releasing hormone ethylamide in normal men. Luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T) responses to three consecutive intravenous injections of D-Leu-6-LH-releasing hormone ethylamide (D-Leu-6-LH-RH-EA) at 3-hour intervals were studied in six healthy, fertile, male volunteers 34.2+/-1.6 years of age. Each man received three injections of 20 microng of D-Leu-6-LH-RH-EA at 6:00 A.M., 9:00 A.M., and 12:00 noon, respectively. Blood samples were obtained before the first injection and at 1, 2, 3 (before the second injection), 4, 5, 6 (before the third injection), 7, 8 and 9 hours after the beginning of the test. Serum levels of LH, FSH, and T were determined by radioimmunoassay with the double-antibody technique. The response to the first injection of D-Leu-6-LH-RH-EA confirmed the longer duration of the stimulation of LH and FSH release caused by this compound as compared with that caused by LH-RH. Serum T levels rose significantly, almost paralleling the variations experienced with gonadotropins. The second injection caused a slight increase in LH and T responses in relation to the first injection. Two and three hours after administration, the third stimulus resulted in hormone levels lower than those obtained with the first two injections. Possible explanations for this finding might be a reduction of pituitary responsiveness as a result of multiple stimulation with D-Leu-6-LH-RH-EA, or spontaneous circadian variation of the pituitary response, or a combination of factors.


Volume and shape of normal human spermatozoa. An improved apparatus for measuring the electrical size of particles, developed in this laboratory and based on the principle of the Coulter counter, is used to size human spermatozoa. The typical size distribution is unimodel, with a skew to the right. The actual quantity determined by the measuring system is electrical size (i.e., shape factor X volume); in order to extract the volume, it is necessary to obtain an independence measure of particle shape. This is done by estimating the relative contributions of each part of the spermatozoon, and gives a weighted value for the shape factor of 1.28. The mean value of spermatozoa from 25 normal human seminal fluid specimens is found to be 17.4+/-1.46 cu micronm and the modal volume, 15.2+/-1.27 cu micronm. These values are compared with data reported in the literature after correcting the latter for the effects of particle shape. Zaponin does not affect the cell volume distributions, even when used in high concentrations, provided measurements are carried out within the hour.


Postcoital contraceptive effects of agonist analogs of luteinizing hormone-releasing hormone. Various analogs of synthetic hypothalamic luteinizing hormone-releasing hormone (LH-RH) were evaluated for agonistic (ovulation-inducing), postcoital contraceptive, and direct uterotrophic activities. All analogs showing agonistic activity also possessed the ability to terminate pregnancy, as did LH-RH; there appeared to be a direct relationship between agonistic and postcoital potency and activity. The highly potent and active LH-RH agonist, D-[Ala]6-des-[Gly]10-pro9-ethylamide-LH-RH, proved to be the most potent and active postcoital preimplantational and postimplantational antifertility agent. In contrast to LH-RH, none of the analogs tested in the hypophysectomized animal produced a uterotrophic effect, revealing a selective extrapituitary effect of the parent hormone. The collective data demonstrate that peptides derived from LH-RH and bearing agonistic properties can terminate pregnancy postcoitally, via disruption of the pituitary-ovarian reproductive complex. Possible mechanisms are discussed, and the use of members of this neurohormonal class as potential profertility agents should be weighed with caution.


Specific suppressor cells in rats with neonatally induced transplantation tolerance. The presence of suppressor cells was investigated in AVN rats bearing Lewis skin grafts of long standing after neonatal induction of transplantation tolerance. Pooled spleen cells from tolerant rats transferred to sublethally irradiated syngeneic recipients prolonged significantly and specifically the survival of skin allografts. Also, the factor present in the medium after cocultivation of lymphocytes from tolerant rats with Lewis antigens was effective.


Strong histocompatibility antigens in Urodela amphibians. In 13 experimental groups skin allo- and xenografts were performed from one donor to several hosts of Tirturus alpestris and T. vulgaris. In all but two groups chronic rejection reaction was observed. Extraordinarily quick rejection (MST = 8 days) in the remaining two groups may be explained by the presence of strong histocompatibility antigens.


Preprosthetic surgery.

Preprosthetic surgery. Several of the abnormal conditions existing in the edentulous patient can be corrected surgically, prior to construction of dentures, to enable the patient to function more successfully following prosthetic restoration. The more common problems have been discussed which often require surgical correction. In some instances, articulated diagnostic models of the patient's mouth show that surgery can be avoided, or when surgery is necessary, these models help identify the exact locations of the tissue to be corrected. Alveolectomy has been presented with an eye toward conservatism since this procedure affects the quality of denture foundation and therefore denture stability. Alveolectomy should be performed only when there is a definite indication for the procedure. Correction of soft tissue abnormalities can substantially improve the patient's ability to function with dentures and solve many of the adjustment problems which confront the dentist. It is often better to correct the soft tissue abnormality rather than to circumvent the problem with an inferior prosthetic restoration. Since the introduction of resilient acrylics and tissue conditioning materials, the patient need no longer suffer the discomfort of denture withdrawal after surgery. Electrosurgery has emerged as a successful method of correcting many soft tissue abnormalities. Vestibuloplasty is receiving much emphasis as a possible treatment for atrophic ridges in which there is still enough alveolar bone from which to extend a sulcus. These sulcus extension procedures may offer some help in the future regarding the problem of resorbed ridges, but presently, more research is necessary before widespread use is recommended.


Complete denture occlusion.

Complete denture occlusion. The occlusion for complete dentures discussed in this article is based on basic principles and concepts that have been studied, tested, refined, and applied successfully by dentists for years. This should simplify rather than complicate the selection of the proper posterior tooth form for each patient's edentulous condition, the the modification of the teeth, and the use of the many occlusal schemes. The problem of occlusion is the dentist's responsibility and cannot be shared or relegated to nonprofessionals. The motivation for this article has been the hope that it will help the dentist recognize, analyze, and apply sound procedures in managing the problem. Only after this has been skillfully accomplished has the dentist assumed his full professional responsibility to the patient.


The trial denture.

The trial denture. 1. Many difficult situations can be avoided if the dentist keeps constantly aware of the physiologic, the psychologic, and the artistic factors involved in the construction of dentures, whether they be partial or complete. 2. No one would think of buying an automobile without getting adequate instruction in how to drive; neither should a patient expect to wear artifical restorations without training and practice. 3. The dentist at the try-in should check: a. The bases for fit and extension. b. Thin labial flange at frenum area. c. Posterior palatal seal and length of upper. d. Posterior tooth poisition for height of plane, relation of plane to alatragus line, tongue room, and ridge relation. e. Anterior tooth position for lip support, length, and relation to lower lip. f. Entire setup for vertical dimension of the face, test balance and simultaneous tooth contacts in all desired positions. If discrepancies exist between the articulator and the mouth, make new jaw relation records. g. Check palatal contour and incisor positions in speaking. "F" and "V" sounds to test the upper incisor, sibilant sounds for vertical dimension and lower-to-upper incisal relationships. h. Wax-up for support and proper external form. If tori exist on upper or lower, be sure there is sufficient thickness to allow relief later on. i. Be sure to repeat and continue instruction of the patient in his understanding and handling of the new dentures. Now, in addition to all these checks to dentist makes, he must constantly anticipate the thoughts and attitudes of the patient and interpret them for better rapport and ultimate patient understanding. No stage in complete dentures is anticipated with such mixed emotions as is the try-in. No stage is so significant to the future success or failure of the patient to take dentures in his stride. No stage has so much potential for making the person happy or sinking him into the depths of despair. The return of self-confidence, the pride in appearance, the sparkle and drive that return to a patient when the restorations are enthusiastically accepted are a wonderful reward for dentistry well done.


Processing complete dentures.

Processing complete dentures. The processing of acrylic resin dentures is an exacting art but is relatively simple to carry out to perfection day after day, even when several dentures are processed at the same time. Nine salient factors in the proper processing of acrylic resin dentures were sequentially listed. Unfortunately, a number of these technical procedures are frequently overlooked or shortcuts may be substituted. Herein lie the causes of some hitherto unexplained clinical failures attributed to the acrylic resin denture base material. The acrylic resin denture base materials have been and are currently the best plastic for making dentures. This material has many unique assets and few faults. Its fine properties are often overlooked by the dentist who has several edentulous patients experiencing problems with dentures he made for them. Sore spots, faulty occlusion, looseness, and even breakage are most likely due to inadequate mouth conditioning or poor impressions, incorrect jaw relations, or the dentist's failure to remount the dentures prior to insertion. We have sufficient knowledge and scientific evidence to realize that most, if not all, denture problems are caused by the dentist's technique or by dentist-patient relationship. These problems should not be attributed to the acrylic resin material nor to the hard-working, conscientious dental laboratory technician. I doubt that more than 2 per cent of all complete denture difficulties experienced by frustrated patients would be solved by the introduction and availability of a new perfect denture base material.


Obturators on complete dentures.

Obturators on complete dentures. A philosophy and technique for the preparation of removable silicone rubber obturators on complete dentures have been presented. Either of two methods for construction is used depending on whether the defect is large or small. Advantages of the silicone rubber obturator-denture combination were elaborated.


Implants for fixed and removable prostheses. For those individuals whose oral anatomy contraindicates the use of endosteal implants, there still remains a viable alternative to conventional removable dentures. Both maxillary and mandibular implant dentures of a subperiosteal design will provide a stable implant and a retentive restorative prosthesis. The patient is free from the oral gymnastics required to manipulate such appliances and in the maxilla the dentist is able to eliminate all palatal coverage without compromising the retention of the bridge. The lower subperiosteal implant and the pterygoid extension implant discussed both obtain their stability from accurate fit of the implant substructure to the bone. The implant first becomes more retentive when the periosteum tenaciously reattaches itself to the bone and in turn secures the implant in position (Fig. 13). The restorative procedures follow sound prosthetic prinicples with particular attention on avoiding any impingement of the underlying tissue areas. Implant dentures have truly provided patients a means of exploring alternative treatment modalities other than conventional removable dentures. Literally thousands of patients today are living testimony to the accomplishments of oral implantology when pursued as an exacting science prescribed by colleagues experienced in this revolutionary dental discipline.


Species variation in pancreatic islet monoamine uptake and action. There are conflicting data in the literature regarding the role of monamines in the secretion of insulin. In order to clarify the contribution that species variation may make to these divergent results, the uptake of serotinin (5-HT), dopamine (DA), and their precursor amino acids, 5-hydroxytryptophan (5-HTP) and L-dopa, into islets was studied. Islets from golden hamsters, rabbits, guinea pigs, and obese, hyperglycemic mice were isolated by the collagenase technique. The islets were incubated in Krebs-Ringer buffer in the presence of 14C-labeled monamines or their precursors. At 30-minute intervals after initiating the study, the incubation mixture was passed through a Millipore filter. The retained islets were disrupted by sonication and the radioactivity counted. The ratio of the uptake of 5-HTP to 5-HT was at least 3:1 in the hamster, guinea pig, and mouse. In the rabbit the ratio was 1:1. A similar relationship was noted for the uptake of L-dopa and DA. The in-vitro results were confirmed by the in-vivo studies, in which hamsters were injected with 14C5-HT or 5-HTP, followed by isolation of the islets. We conclude that there is significant species variation inthe uptake of these monoamines and their precursors.


Potentiation of hypoglycemic effect of chlorpropamide and phenfromin by halofenate. The potentiation of oral hypoglycemic drugs by the antilipemic agent halofenate is reported. Forty-seven diabetic patients were treated for 48 weeks with halofenate, clofibrate, or placebo. Five patients in the halofenate group were taking phenformin plus either chlorpropamide or tolbutamide. Their average initial fasting plasma glucose was 160 mg./dl. All five patients experienced a slow but but substantial fall in fasting plasma glucose. The mean fasting plasma glucose for the five patients after 80 days of halofenate treatment was 63 mg./dl. As oral treatment for diabetes was reduced, the fasting plasma glucose returned to prehalofenate levels. In this study, we did ont detect an effect of halofenate on the fasting plasma glucose of diabetic patients treated with insulin or on the fasting plasma glucose levels of patients treated with diet alone.


Gastrointestinal cancer studies in the human to nude mouse heterotransplant system. Human gastrointestinal cancer xenografts were established in the nude mouse. Grafts were accomplished with gastric adenocarcinomas, gastric leiomyosarcoma, histiocytic lymphoma of the stomach and gallbladder, pancreatic tumors, colonic cancers and cell lines of duodenal (HUTU-80) and pancreatic (HS-766-T) cancers, melanoma (SK-Mel-5), and murine metastasizing Lewis lung carcinoma. The rate of successful xenografting of these tumors varied from virtually 100% with colon and duodenal cancer, 50% for a pancreatic cancer (P-1), to only 17% for gastric adenocarcinoma. Pancreas and colon adenocarcinomas have been maintained by successive xenotransplantation over 16 and 19 months, respectively. Human xenografts retained morphological identity with tissues of origin through several transplant generations and shared some of their ultrastructural characteristics but did not metastasize. Rodent xenografts, of heterogenous origin were characterized by differences in the duration of the latent period and in the rate of their initial development as described by the average doubling times and average slopes (B) of their growth curves. Differences between B of the Lewis lung carcinoma and all of the human xenografts and between B of a pancreatic adenocarcinoma and three other neoplasms were significant (P less than 0.05 to 0.04). Labeling indices determined for 14 cancer transplants were in the range of previously reported data for similar neoplasms in patients or other xenograft systems. These findings suggest that the nude mouse model can be used to evaluate endogenous properties of gastrointestinal cancers and their responses to exogenous agents.


Septicemia as a complication of percutaneous liver biopsy. Three cases of septicemic shock after liver biopsy are reported. Escherichia coli was cultured in 2 patients and in 1 E. coli was cultured with Streptococcus viridans. All 3 patients were afebrile and had normal WBC counts before the procedure. Two had cholangitis and the 3rd had primary hepatoma and cirrhosis of the liver. Septicemic shock should be recognized as a rare complication of liver biopsy in patients with biliary obstruction.


Stimulation of L-asparaginase production in Escherichia coli by organic and amino acids. The effect of 18 amino acids and 7 organic acids on the production of L-asparaginase EC-2 by a strain of Escherichia coli in a chemically defined medium was investigated under moderate aeration. All the amino acids and some of the organic acids stimulated the enzyme production. The specific activity without stimulants was about 0.16 nkat per mg dry weight, with stimulants it lay between 1 and 6 nkat per mg dry weight but with L-leucine and L-methionine the values were 12 nkat and 17 nkat per mg, respectively. When two organic or amino acids were added simultaneously at concentrations that were suboptimal for stimulation, the stimulating effects were cumulative in most cases. When cells were grown under conditions approaching anaerobiosis, the specific activity reached, even in the absence of stimulants, values as high as 5 nkat per mg; under these conditions, a further substantial increase in specific activity was only caused by L-leucine and L-methionine. Stimulating effects of DL-lactate and of some amino acids were also found in other strains of Escherichia coli. The ability to grow on a medium with L-asparagine as the sole source of both nitrogen and carbon was found in two strains; growth took place even when there was no measurable activity of L-asparaginase EC-2.


Physical properties of microbial suspensions. II. Properties of microbial suspensions and their supernatants during fermentation conditions. Physical properties of suspensions of Saccharomyces cerevisiae, Candida utilis and Escherichia coli (density, viscosity and surface tension) were measured in synthetic suspensions formed of centrifuged biomass and supernatants from various stages of batch cultivation in the range from 0 to 10% w/v for yeasts and from 0 to 0.25% w/v for bacteria. Surface tension was also measured in native suspensions in the range of 0 less than or equal to Cm less than or equal to 2.0% w/v. All single cell suspensions were found to be Newtonian in behaviour. Densities strictly obey the mixing law, viscosities of Saccharomyces cerevisiae suspensions were correlated by an empirical relation in dependence of Cm and t, surface tensions were correlated graphically for suspensions of Saccharomyces cerevisiae and Escherichia coli, since experiments with both microorganisms have shown that the previously published approximate correlation can safely be used.


[Determination of the viability of skin regions in danger of necrosis by means of transcutaneous polarographic measurement of oxygen pressure]. In 8 patients the method of transcutaneous pO2-monitoring, developed by Huch and Lübbers, is applied for the determination of the viability of ischemic skin areas of the transplant. Its usefulness is proven. The problem of arterial and tissue pO2-monitoring is explained. According to our experiences of about 6 months this qualitative method seems to be useful in plastic surgery. An important possibility to apply the transcutaneous pO2-monitoring is the postoperative control of free skin flaps transplanted by microvascular anastomoses to indicate early enough the beginning tissue hypoxia by a warning system. Further results with the method shall be reported.


Repair, replication and survival in uv-irradiated Escherichia coli. The influence of dimer removal through excision or photoreactivation on the kinetics of DNA synthesis, sedimentation profiles of DNA molecules and survival of cells was investigated in excision-deficient and excision-proficient Escherichia coli K-12 after a flux of 20 J M-2. In excision-deficient cells photoreactivation did not influence the kinetics of DNA synthesis for a long period and the sedimentation properties of DNA synthesized immediately after photoreactivation were influenced only slightly. However, survival was increased remarkably. In excision-proficient cells where dimers were removed through excision, the kinetics of DNA synthesis increased rapidly, normal-sized DNA molecules were synthesized 60 min after irradiation and survival was substantially higher than in the above-mentioned case. This can hardly be interpreted as a more complete repair of dimers by excision because the persistence of dimers in these cells did not significantly influence either the kinetics of DNA synthesis or normalization of DNA molecules and/or survival of cells. It is concluded that persisting dimers play an important role in excision-deficient but not in excision-proficient cells, that a non-dimer damage to DNA causes inhibition of DNA synthesis after UV and that this damage is of primary importance for excision-proficient cells which can easily cope with persisting dimers.


[Electronic data processing in the clinical nuclear medicine]. Computer techniques as introduced into clinical nuclear-medicine 12 years ago, were first exclusively used for scintigraphic purposes. Through the development of a comprehensive pilot system for nuclear medical data processing on the basis of two Siemens Process control computers it could be demonstrated, that besides of scintigraphy there are also other important fields for computer application, e.g. in function diagnostics and therapy planning.


Prolonged intravenous infusions of LH-releasing hormone into normal men. Five normal men received constant intravenous infusions of luteinizing hormone-releasing hormone (LH-RH), 0.2 mug/min, for 14-19 hours. Serum levels ofluteizining hormone (LH) revealed a biphasic pattern of increase, reaching maximal values by 4 hours after the infusions began, then remained near that level until the infusions ceased. Serum follicle stimulating hormone (FSH) levels rose gradually to maximal values by 6-13 hours and maintained this level until the end of the infusions. Testosterone (T) levels revealed gradual increases throughout the infusions. These results confirm an increase in serum T levels with prolonged endogenous gonadotrophin stimulation. This is in contrast to the inability of several previous studies to demonstrate an increase in T levels following the relatively short gonadotrophin elevation produced by single-shot LH-RH administration. The T increases produced, however, were quantitatively much less than those reported during prolonged LH-RH infusions in rams, suggesting that the human testis is less responsive to endogenous gonadotrophin stimulation than is that of the ram. In addition, prolonged LH-RH stimulation did not cause pituitary refractoriness in men as has been described in animals.


Factors influencing insulin and glucagon secretion in lean and genetically obese mice. The control of insulin and glucagon secretion from isolated pancreatic islets of lean and genetically obese mice has been compared. The enlarged islets of obese mouse pancreas and islets of obese mouse pancreas and islets of obese mice maintained on a restricted diet manifested a greater response to glucose stimulation of insulin secretion than the lean mice islets. The glucagon content of the islets, the secretion of glucagon in a medium containing 150 mg% glucose and the stimulation of glucagon secretion by arginine did not differ significantly in the two groups. Adrenaline stimulated glucagon secretion in vitro from obese mice but not from lean mice. Antinsulin serum injections into obese mice increased the plasma glucagon levels about twofold and had no effect on glucagon levels in lean mice, although the level of hyperglycaemia was the same in both groups. It is suggested that the suppression of glucagon release by glucose requires a higher concentration of insulin in the obese mouse pancreas than in lean mice.


Immunocytochemical evidence for glucagon-containing cells in the human stomach. To determine if glucagon-containing cells could be identified in the human fundus, stomachs attained at autopsy within 4-hours of death from persons previously considered to be in good health were examined by the indirect immunoperoxidase technique using antiglucagon serum 30K. Glucagon-containing cells were demonstrated in one of eight gastric fundi examined. The glucagon content of acid alcohol extracts of the fundi examined. The glucagon content of acid alcohol extracts of the funci was low in all cases. Glucagon content was also low in canine stomach removed 4-hours after death. It is concluded that glucagon-containing cells, demonstrable by immunocytochemical techniques, may be present in the gastric fundus of humans.


Immunochemical studies on phenylalanyl-tRNA synthetase from Escherichia coli. Antibodies against the alpha and beta subunits of phenylalanyl-tRNA synthetase were fractionated by ion exchange chromatography into different classes and then digested with papain to yield the respective Fab fragments. The preparations obtained were used to investigate (i) whether the alpha and beta polypeptides share any common antigenic determinants and (ii) whether immunological methods are able to resolve the catalytic function of the subunits of this enzyme (or principally of oligomeric enzymes). As to the first problem, immunodiffusion and complement fixation experiments showed that there is no immunological relatedness between the subunits which argues against the existence of sequence homoligies. As to the second question investigated, it was found that any binding of immunoglobulins of Fab fragments to the alpha or the the beta subunit affects enzyme activity either in the direction of activation or inhibition. These results therefore show that the immunological approach is not appropriate for resolving subunit-specific funcitons, possibly as a consequence of conformational changes induced in the enzyme by the binding of the immunoglobulins of Fab fragments.


Karyotype peculiarities of malignant lymphomas. Karyotypes of 30 malignant lymphomas were studied with the aid of G-banding. Frequent occurrence of rearranged chromosomes 14 and 11 was noted. In several tumors, identical acrocentric markers, appearing after translocation of the long arm of chromosome 11 on the long arm of chromosome 14, were revealed. Other common karyotype abnormalities in lymphomas were trisomy 3 and trisomy 18. The chromosomes preferentially involved in karyotype abnormalities of the malignant lymphomas are mostly not altered in other hemoblastoses.


[Heterogeneity of antinuclear antibodies (ANA), immunoglobulin- and complement levels in sera of patients with rheumatoid arthritis (RA) (author's transl)]. ANA typing of RA patients according to the heavy- and light chains, complement fixing ability and immunofluorescent staining pattern was performed by the immunofluorescence technique and partially by qualitative immunoelectrophoresis. In the corresponding sera the immunoglobulins and complement components were quantiatively determined. Dependent on the ANA-immunoglobulin classes we found the following distribution: IgG-ANA; 40/93 (43%), IgM-ANA: 19/93 (20%), IgA-ANA: 2/93 (2%), and Ig-comb.-ANA:17/93 (18%). ANA only positive for polyspecific antimmunoglobulin serum: 15/93 (16%). After L-chain typing we found within a given ANA-Ig-class positivity for both L-chain subclasses (kappa and lambda): IgG-ANA: 19/40 (48%), IgM-ANA: 3/19 (16%), IgA-ANA:1/1 and Ig-comb.-ANA: 14/17 (82%). ANA cases within a given Ig-class only positive for the L-chain/type kappa:IgM-ANA:6/19 (32%), IgG-ANA: 9/40 (23%), Ig-comb.-ANA:2/12 (12%). ANA cases within a given Ig-class only positive for the L-chain type lambda: IgM-ANA: 7/19 (37%), IgG-ANA: 3/40 (8%), Ig-comb.-ANA:1/17 (6%) and IgA-ANA:1/1. In all cases of restricted ANA-positivity (only positive by use of polyspecific anti-immunoglobulin serum) no light chains were detected. In total we found approximatively 25% of complement fixing ANA, predominantly IgG-ANA (70%). Concerning the immunofluorescent nuclear staining we stated the following patterns: homogenous: 34/93 (36.6%), speckled: 29/93 (31.2%), mixed pattern : 22/93 (23.6%) peripheral: 5/93 (5.4%) and nucleolar: 3/93 (3.2%). The quantitative Ig-determination reveals a significant increase of IgG in ANA (single type) positive sera. IgG + IGM + IGA-ANA and IgG + IgM-ANA are found together with a significantly increased IgG- and IgM-serum levels, in the case of IgG + IgM-ANA we could demonstrate an additional relative decrease of the complement component C4. Sera with positive complement fixing ANA compared with non complement fixing ANA are characterized by an absolute increase of IgG and relative decrease of the complement component C4. No differences in serum-immunoglobulin- and -complement concentrations were registered dependent on ANA-immunofluorescent staining patterns.


[Clinical importance of the complement system (author's transl)]. It is intended to summarize the latest research in the complement field. After a short comment on the biochemical behaviour of proteins participating in the complement reaction, biochemistry of the complement reaction itself and its biological activities are described. The question of participation of the complement system in disease is examined by specifying the possibilities to analyse the complement system in clinical problems, and, on the other hand, by giving a review on our latest knowledge of modifications of the complement system in disease.


Involvement of T cells in the recall of Salmonella-induced resistance to tumour. Immunization of mice with Salmonella enteritidis 11RX (11RX) induces resistance to tumour growth in these animals. The degree and duration of resistance is dependent on the routes used for immunization and tumour challenge. When resistance to tumour growth has declined, it can be recalled using various 11RX antigen preparations. In the present study we have shown that the ability to recall resistance to i.p. challenge with 131I-labelled Ehrlich ascites tumour cells could be transferred to normal mice by i.p. injection of an antigen preparation of 11RX mixed with spleen cells from 11RX-immunized mice. The active cells in this test were apparently sensitized T cells, since spleen cells from normal mice were not effective and the activity of immune spleen cells could be markedly reduced by removing theta antigen-bearing cells but not by removing glass-adherent cells. The induction of in vivo cytotoxicity appeared to depend on the involvement of radiation-sensitive cells derived from the non-immunized recipients, since recall of resistance to tumour challenge was reduced when irradiated mice were used as recipients of spleen cells from immunized mice.


Physiochemical consequences of opsonization of Salmonella typhimurium with hyperimmune IgG and complement. Partition in an aqueous, two-polymer phase system containing dextran and polyethylene glycol was employed to investigate the physico-chemical changes inflicted upon the cell surface of a smooth strain of Salmonella typhimurium by the binding of antibody IgG and complement. The minimum antibody concentration for increased phagocytosis in vitro was approximately the same as that for a significant change in two-phase partition, ca 8000 mol/bacterium, whereas a lower concentration, less than 4000 mol/bacterium, was sufficient to increase clearance in vivo. After pepsin digestion of IgG, larger quantities, ca 35,000 mol/bacterium, was required for opsonization and to influence two-phase partition. Addition of normal rabbit or guinea-pig serum to bacteria sensitized with a low concentration of antibody IgG conspicuously enhanced phagocytosis and affinity for the dextran-rich phase. The results show that binding of 8000 IgG antibody molecules or more to smooth S. typhimurium generates physicochemical changes of the bacterial surface which from studies on S leads to R mutations are known to correlate with hydrophobicity, negative charge and phagocytosis. Such results support the view that one important function of IgG antibody and complement is to decrease the hydrophilic properties of the bacteria which is thought to be a prerequisite for phagocytosis.


Effect of human monocytes and macrophages on Trypanosoma cruzi. Studies were undertaken to determine whether Trypanosoma cruzi can invade and multiply within human monocytes and macrophages cultured in vitro and, if so, whether macrophages can be activated to inhibit the multiplication. A reticulotropic strain of T. cruzi was capable of infecting human monocytes and monocyte-derived macrophages. Intracellular multiplication was observed in both cell types when they were examined microscopically. An increase in the number of trypanosomes occurred in the supernatants as well, providing additional evidence of intracellular multiplication and cell disruption by the parasite. Activation of monocyte-derived macrophages was accomplished by incubating the monolayers in the presence of lymphocytes and streptokinase-streptodornase. These activated macrophages inhibited intracellular multiplication of T. cruzi and the number of T. cruzi in the supernatants of these monolayers was markedly decreased as well.


Mechanism of formation of non-immune rosettes between guinea-pig thymus-derived lymphocytes and rabbit erythrocytes. Several observations reported here suggest that spontaneous rosette formation between rabbit erythrocytes and guinea-pig T lymphocytes is mediated by natural anti-guinea-pig T-cell antibodies bound to the surface of the rabbit erythrocyte. First, normal rabbit sera frequently contain antibodies specifically cytotoxic for guinea-pig T lymphocytes. Second, the activity of rabbit erythrocytes in spontaneous rosette formation is reduced after incubation for 5 days at pH 6-1, but can be restored to levels seen with fresh erythrocytes by a brief incubation in normal rabbit serum containing natural anti-guinea-pig antibodies; normal serum absorbed with thymocytes does not restore activity to the erythrocytes. Third, the activity of rabbit erythrocytes in forming spontaneous rosettes can be specifically blocked by treatment with anti-allotype and heterologous anti-Ig sera.


A comparative study on the humoral immune responses in germ-free and conventional mice. The plaque-forming cell (PFC) responses to sheep red blood cell (SRBC) dinitrophenyl-lysine-Ficoll (DNP-lys-Ficoll), and dinitrophenylated bovine serum albumin (DNP-BSA) have been studied in both germ-free and conventionally reared ICR mice. In germ-free mice, the IgG response to SRBC and the IgM and IgG responses to DNP-BSA were lower than in conventional mice, but no difference was observed in the IgM response to SRBC or the IgM and IgG responses to DNP-lys-Ficoll. Further, the number of 0-bearing cells in the spleen was smaller, and the mitogenic response of spleen cells to PHA was lower in germ-free mice than in conventional mice. These observations suggest that T cells of germ-free mice remain functionally immature.


Effect of an acidic polysaccharide produced by Serratia piscatorum on immune responses im mice II. Stimulatory effects in normal and immunologically impaired animals. An acidic polysaccharide (PS) of Serratia piscatorum enhances the IgM PFC responses against heterologous erythrocytes in mice. Early and late IgM responses were increased significantly by increasing the number of immunizing erythrocytes and the dose of PS, whereas the IgM PFC response was suppressed by higher dose of PS and antigen. A stimulatory doses of PS significantly increased the secondary IgM and IgG responses against sheep erythrocytes. PS restored the reduced PFC response against sheep erythrocytes in adult-thymectomized, 60Co-irradiated and bone marrow-transferred mice (ATXBM) and nude mice (nu/nu), and thus the stimulatory effect of PS appeared greater in immunologically impaired mice than in normal ones. Spleen cells taken at the time of the peak PFC response from mice treated with higher doses of sheep erythrocytes and PS, suppressed the primary IgM production of normal syngeneic spleen cells against sheep erythrocytes in vitro. The suppressing activity of the spleen cells was increased by prior treatment with anti-theta serum and complement, while it was reduced by treatment with anti-mouse Ig serum and complement. These results suggested that immunoglobulin-bearing cells may have a role on the suppressing activity of spleen cells.


The effect of trypsin and chymotrypsin on the bactericidal activity and specific antibody activity of bovine colostrum. Digestion of bovine colostral whey with trypsin or chymotrypsin caused a progressive loss of the complement-mediated bactericidal activity of naturally-occurring colostral antibodies of E. coli 0111. Bactericidal activity was associated primarily with IgG1 immunoglobulin and to a lesser extent with IgM. Chymotrypsin preferentially attacked IgM, destroying its antibacterial activity and producing an apparent decrease in its mol wt. Trypsin preferentially attacked IgG1, but loss of antibacterial activity was in this case not accompanied by a decrease in molecular weight. Using colostral whey with antiperoxidase activity it was shown that  the kinetics of loss of specific antibody activity were similar to those of loss of bactericidal activity. It is therefore suggested that trypsin may cause a loss of specific antibody activity of colostral IgG1 without cleaving the immunoglobulin molecule.


The effect of limited proteolysis by trypsin and chymotrypsin on bovine colostral IgG1. Limited proteolysis of bovine colostral IgG1 by trypsin caused loss of specific antibody activity but column chromatography showed that relatively little cleavage into fragements had occurred. Polyacrlamide-agarose SDS electrophoresis of the 2-mercaptoethanol-treated digest revealed, however, that extensive cleavage of light chains had occurred even though most of the material before reduction had a mol. wt close to that of undigested IgG1. Although a Fab-type fragment was detected in the digest by immunoelectrophoresis it appeared to be only a minor component. Chymotrypsin had little effect upon either the structure or antibody activity of IgG1. These findings may explain the effect of trypsin and chymotrypsin on the bactericidal activity of colostral antibodies.


Receptors for IgM on murine lymphoid cells. Murine lymphoid cells from the thymus, lymph node and spleen were shown by indirect immunofluorescence, to bear membrane receptors for purified IgM of human origin. The bond between IgM and the cells took place through the Fc re gion, as demonstrated with isolated fragments of the normal IgM molecule. Two distinct myeloma IgM lacked this biological activity. The cytophilic origin of part of the immunoglobulin usually described on T-cell membranes is discussed.


Cell-mediated and humoral immune responses in mice. IV. Difference of the functional cell population between helper activity and delayed-type hypersensitivity. Helper activity in the anti-hapten antibody response was studied in mice in reference to the induction of delayed-type hypersensitivity (DTH) to the carrier protein. Mice were immunized either by an i.v. injection of alum-precipitated bovine serum albumin (AP-BSA) plus bacterial endotoxin or by a s.c. injection of BSA in Freund's complete adjuvant, the latter being effective in inducing DTH. The helper activity was estimated by the antibody response to the challenge with dinitrophenylated BSA (DNP-BSA) given at varying intervals after the injection of BSA. The results indicated that the helper activity was independent of DTH to the carrier protein, suggesting that these two activities, are mediated by different populations of functional cells. A low dose of tolerogenic soluble BSA (sBSA) was sufficient to abrogate the helper activity in the response to DNP-BSA. In contrast, DTH to BSA was only partially depressed by the pretreatment with a low dose of sBSA and was completely depressed by a high dose. DTH reactivity in mice pretreated with a low dose of tolerogen and followed by the immunization with BSA in Freund's complete adjuvant was substantiated by the microscopic observation of mononuclear cell infiltration at the site of the test antigen injection. These results suggest that cells involved in the helper function and DTH may be derived from different precursors.


The effects of synthetic polymeric agents on immune responses of nude mice. The influence of synthetic polymeric agents on the immune responsiveness of congenitally athymic (nude) mice was investigated by determining the effects of in vivo treatment with polynucleotides and polymeric haptenated antigens on splenic theta-bearing cells, on mitogen stimulation and on plaque-forming cell responses to thymic dependent and thymic independent antigens. Contrary to in vitro data, no evidence was obtained to demonstrate in vivo restoration of these immune parameters by the use of non-specific immune enhancers. Further, despite the continued release of lipopolysaccharide from the bowel, older nude mice (10 months old) demonstrated no acquisition of improved T-cell function. Nude mice responded well to the thymic independent antigen p-azobenzenearsonate-N-acetyl-tyrosylglycylglycine (A-TGG) Ficoll. Finally, the class specific responses to the thymic independent antigen DNP-Ficoll were significantly different from those of nu/+ littermate controls, indicating the importance of thymic influences upon the class switching of immunoglobulin responses.


Proteolysis of lymphocytic surface immunoglobulin. Limited proteolysis of lymphocytic surface immunoglobulins in guinea-pig, rabbit and man was investigated by immunofluorescence using conjugated antisera specific for immunoglobulin fragments. The cell surface IgM of guinea pig L2C leukaemic lymphocytes and rabbit blood lymphocytes was cleaved in situ at its hinge region by papain. The Fcmicron fragment remained attached to the membrane and could be stained with the appropriate anti-Fc conjugate. The surface IgD and IgM of human chronic lymphocytic leukaemia cells was cleared from the cell surface by papain, as shown by reagents directed against both Fab and Fc region determinants. This could be due either to proteolytic degradation of membrane bound Fc or to initial cleavage of Ig from the membrane at some point other than the hinge region.


Cell-mediated immunity in experimental Nocardia asteroides infection. Experimental mycetoma-like lesions developed in guinea pigs after subcutaneous injection of Nocardia asteroides. Although delayed hypersensitivity appeared earlier, increased macrophage migration inhibition and microbicidal activity appeared after 7 weeks. When the lesions healed, high cell-mediated immunity was present. Cell-mediated immunity was transferred to normal recipient guinea pigs from healed donor guinea pigs by spleen cell transfer. Recipient guinea pigs showed marked protection against challenge with N. asteroides.


In vivo and in vitro cell-mediated immune responses to a cell wall antigen of Blastomyces dermatitidis. An alkali-soluble, water-soluble cell wall fraction of Blastomyces dermatitidis, designated B-ASWS, was evaluated as an antigen for detecting in vivo (skin tests) and in vitro migration inhibition factor (MIF) production and lymphocyte transformation (LT) responses in Blastomyces-infected guinea pigs. The biological activity of B-ASWS was compared with that of blastomycin KCB-26. The superiority of B-ASWS, in terms of its sensitivity and specificity, was evident in in vivo and in vitro assays. Skin tests responses were obtained in 21 of the 24 Blastomyces-infected guinea pigs, whereas only one of the 14 Histoplasma-infected guinea pigs were significantly greater than those obtained using cell populations from Histoplasma-infected or noninfected guinea pigs. The con-MIF and LT in peritoneal exudate cells and lymph node cells of homologuosly infected animals. In each biological system, the response of the Blastomyces-infected guinea pigs were significantly greater than those obtained using cell populations from Histoplasma-infected or non-infected guinea pigs. The contrasting efficacy of B-ASWS as compared with blastomycin KCB-26, suggests that the cell wall antigen will be a useful tool for detecting cell-mediated immune responses in blastomycosis.


Interaction of Treponema pallidum (Nichols strain) with cultured mammalian cells: effects of oxygen, reducing agents, serum supplements, and different cell types. Cultured mammalian cells extend the time of survival of Treponema pallidum (Nichols strain). Various parameters that have been previously shown to enhance treponemal survival in vitro were examined for influences on the interaction of T. pallidum with cultured cells. With cells derived from normal rabbit testes, the time of retention of treponemal virulence was extended in an atmosphere containing reduced concentrations of oxygen. Glutathione and cysteine, when added to the basal tissue culture medium, prolonged treponemal survival. In an assessment of various tissue culture medium supplements, normal rabbit serum was equivalent to fetal bovine serum and superior to bovine serum albumin fraction V (BSA), fatty acid-poor BSA, and lipid-pooed for TRK-2, HSE, NRK, and C6 cells. Dithiotreitol, as an additional reducing agent, sharply enhanced treponemal survival. With SF1Ep NBL-11 cells and basal tissue culture medium containing glutathione, cysteine, and dithiothreitol, in an atmosphere of approximately 3% oxygen, T. pallidum was maintained without detectable decreases in the number of virulent organisms for 6 days.


Comparison of the virulence of O:9,12 and O:4,5,12 Salmonella typhimurium his+ transductants for mice. The purpose of these experiments was to determine whether qualitative differences in the O repeat unit of the lipopolysaccharide of pathogenic Salmonella species affected virulence for mice. O:4,5,12 and O:9,12 sister his+ transductants were derived from a virulent Salmonella typhimurium. Fermentation markers were introduced by transduction to differentiate these strains, and the strains were used as mixed challenge to CF1 and C57B1/6J mice. Liver and spleen cultures of the mice of various intervals after challenge indicated that the O:4,5,12 member of the nearly isogenic pair regularly multiplied to a greater extent than the O:9,12 after intraperitoneal challenge for both strains of mice. After oral challenge, there was little difference in the amount of multiplication of the O:4,5,12 and O:9,12 strains in CF1 mice, but the O:4,5,12 was the predominant strain recovered in 8 of 10 instances in the C57B1/6J mice.


Hyaluronidase activity of bacteriophages of group A streptococci. A sensitive dye-binding assay was employed to study the hyaluronidase associated with temperate and virulent phages infected group A streptococci. Some enzyme was detectable in each purified phage preparation examined, but differences of several orders of magnitude separated the lower enzyme levels in virulent phages that required the addition of hyaluronidase for plaque formation and the higher levels in temperate phages that did not. Infection by virulent phage A25 was accompanied by the production of levels of hyaluronidase proportionate to the average burst size. Hyaluronidase was produced during infection by temperate phages at a much higher level than could be accounted for by the number of phage particles formed. The major portion of this hyaluronidase was free and apparently unassociated with phage or phage fragments. The phage-associated enzyme was tightly bound but could be released and solubilized by treatment with urea.


Use of fluorescent antibody in studies of immunity to cholera in infant mice. Infant mice 8 days of age were infected orally with virulent, motile, classical or El Tor strains of Vibrio cholerae and with nonmotile mutants of low virulence derived from the same strains. At intervals of 8 and 12 h postinfection, frozen thin sections of the ileum were prepared, stained with fluorescein isothiocyanate-labeled rabbit anti-vibrio antibody, and examined with the fluorescence microscope. The motile organisms were present in larger numbers, especially at 12h, and had penetrated the intervillous spaces and crypts of Lieberkuhn more completely than nonmotile vibrios. Dilution counts were made on various regions of the intestines of infant mice challenged orally 12 h previously with either motile or nonmotile strains of V. cholerae. Greater numbers of organisms were found, especially in the upper intestinal regions, when motile organisms were used. Low numbers of vibrios, limited mostly to the lumen, were seen in the ileum of infant mice infected with motile organisms when the infants were the offspring of mothers that had been immunized with crude flagellar vaccine or a vesicular preparation derived from the vibrio cell surface. The distribution of vibrios in this case was similar to that found in infected infants of unvaccinated mothers challenged with nonmotile organisms. Motility appears to enable the bacteria to better populate the upper regions of the intestinal tract and to avoid the washing effects of secretions and peristalsis. Antibacterial immunity may function, at least in part, by making it impossible for motile vibrios to accomplish this widespread distribution within the ileum.


Serological identification of pig enterotoxigenic Escherichia coli strains not belonging to the classical serotypes. Escherichia coli strains isolated from pigs suspected to have succumbed to E. coli enterotoxicosis and not belonging to the commonly incriminated (classical) serotypes (O8:K87:K88, O45:K88, O138:K81:K88, O141:K85:K88, O147:K89:K88, O149:K91;K88, and O157:K88) were tested for enterotoxigenicity in the ligated gut test (LGT) using pig intestine. Of 202 strains tested, 54 strains belonging to 13 different O groups were positive in the LGT. Four of these strains had K88 antigen and one possessed K99 antigen. The majority of the strains was not agglutinated by any of the standard OK antisera. Four new K antigens ("K200", "K442", "K2346" and "K2347") were provisionally designated. K200 was found in pig enterotoxigenic strains belonging to O group 8 and carrying flagellar antigen H31 and in non-enterotoxigenic non-motile strains of O group 8, as well as in O group 20 strains isolated from calves succumbing to E. coli septicemia in two countries. The provisional antigen K2346 was encountered in 18 enterotoxigenic strains with various O antigens from two countries. It is proposed to include these two K antigens into the international E. coli antigens scheme. Attempts to demonstrate a common antigen in the nonclassical enterotoxigenic strains lacking K88 and K99 antigens failed.


Killing of Escherichia coli by a granulocyte fraction occurs without recognizable ultrastructural alterations in the bacterial envelope, as studied by freeze-fracture electron microscopy. Concentrations of a highly purified rabbit polymorphonuclear leukocyte fraction that rapidly caused irreversible loss of viability of Escherichia coli (S15) but reversible envelope alterations produced no recognizable morphological changes as studied by freeze-fracture electron microscopy. These findings support previous evidence that the killing of certain gram-negative microorganisms by granulocyte fractions occurs with minimal structural or functional disorganization of cytoplasmic and outer membranes.


Occurrence of K99 antigen on Escherichia coli isolated from pigs and colonization of pig ileum by K99+ enterotoxigenic E. coli from calves and pigs. Several strains of enterotoxigenic Escherichia coli (ETEC) isolated from pigs were found to have an antigen (K99) previously reported only on strains of calf and lamb origin and which facilitates intestinal colonization in the latter two species. Several human ETEC were also tested for K99; however, none were positive. Each of four K99-positive ETEC strains of calf origin and one of pig origin produced K99 in pig ileum in vivo, adhered to villous epithelium in pig ileum, colonized pig ileum, and caused profuse diarrhea in newborn pigs. In contrast to the K99-positive strains above, four K99-negative ETEC from humans and chickens and one K99-positive ETEC from a calf either did not colonize pig ileum or did so inconsistently. When the K99-negative strains did colonize, they had little or no tendency to adhere to intestinal villi. These results are consistent with the hypothesis that K99 facilitates adhesion to and colonization of pig ileum by some ETEC.


Escape from antibody-mediated immune suppression in vitro by delayed-type hypersensitivity reaction. Anti-sheep erythrocyte antiserum suppressed the in vitro immune response of swine spleen cells to sheep erythrocytes. Inhibition of the in vitro anti-sheep erythrocyte response was also observed in neonatal spleen cells, which were obtained from piglets recently fed colostrum. Addition of purified protein derivative (PPD) to spleen cell cultures prepared from animals previously sensitized to Mycobacterium bovis reversed the immunosuppression caused by passive antibody. Passive antibody regulation was dose dependent; low doses of anti-sheep erythrocyte antiserum caused enhancement, whereas higher doses resulted in complete inhibition of anti-sheep erythrocyte responses. Addition of PPD to cultures of sensitized cells containing passive antibody resulted in progressively increased antibody responses as the dose of anti-sheep erythrocyte antibody increased. PPD addition also reversed maternal antibody suppression of in vitro 2-week-old neonatal spleen cell responses providing these animals were sensitized with M. bovis at the time of birth.


Improved minca medium for the detection of K99 antigen in calf enterotoxigenic strains of Escherichia coli. The K99 antigen of Escherichia coli can be detected more readily in cultures grown on Minca medium at 37 degrees C for 6 to 8 h or grown on Minca plus 1% Iso VitaleX for 20 to 24 h.


Isolation of dinitrophenyl-skin protein conjugates by immunoadsorbent chromatography. Immunoadsorbent columns charged with rabbit anti-dinitrophenyl (DNP) antibody were used to isolate and concentrate DNP-protein conjugates from the 105,000 g supernate of extracts prepared from guinea pig epidermis previously painted with dinitrofluorobenzene (DNFB). Conjugates isolated in this manner elicited positive delayed hypersensitivity skin tests when injected intradermally into DNFB-sensitized animals. Dinitrophenylated epidermal protein conjugates prepared in vitro were capable of specifically inhibiting the migration of peritoneal exudate cells obtained from DNFB-sensitized guinea pigs. These results demonstrate the application of immunoadsorbent chromatography and the migration inhibition assay to the further definition of the epitopes involved in contact sensitivity.


Depletion of IgG in rabbit sera by absorption with some group A streptococci. Streptococci group A, type M 1 or M 56 were capable to deplete IgG in normal rabbit sera during absorption by means of a surface Fc receptor. One absorption with 8.0 mg (dry weight) M 1 streptococci or two absorptions with 71 mg M 56 streptococci removed all IgG in 0.2 ml rabbit serum diluted 1:10 (containing 1.5 mg IgG/ml). Antibodies to egg albumin and antibodies to sheep red cells were not demonstrable after absorption of 0.2 ml serum diluted 1:10 with 16.0 mg M 1 streptococci. These results are discussed in relation to some reports on immunologic cross-reactions between streptococci and mammalian tissues.


Tissue antibodies in malignant and benign urogenital disease. The presence of anti-tissue antibodies in sera from patients with genitourinary carcinomas and non-malignant genitourinary diseases was studied by indirect immunofluorescence. An increased incidence of various tissue antibodies was observed in carcinomas of the urinary bladder, prostate and kidney when compared to controls. There was a trend towards association between antinuclear antibodies and metastatic undifferentiated tumors. A low degree of malignancy was associated with antibodies against connective tissue "reticulin". A striking similarity between tissue antibody "patterns" in carcinoma patients and patients with chronic renal inflammation was seen. This points to inflammation as being an important factor in tissue antibody production.


Characterization of human lymphocyte subpopulations for cytotoxicity against tumor-derived monolayer cultures. Lymphocyte fractions of three healthy donors were tested for cytotoxicity in a 48-h assay on several tumor-derived lines. Analysis of surface markers indicated that a non-T non-B fraction comprising about 50% Fc-receptor-positive cells was most potent. The cell yield in this fraction was on the average 6.5% of the non-fractionated population. Elimination of Slg positive cells did not influence the cytotoxic potential. Pure T cells isolated with E-rosetting subsequent to passage on nylon wool column had no or very low low cytotoxicity. We had indications that cells without conventional markers--"null" cells--were also cytotoxic.


Bullous pemphigoid in childhood: report of three cases and a review of literature. Three proven cases of bullous pemphigoid in childhood which responded to dapsone are reported. The clinical and immunological criteria for diagnosis were similar to those in the aged. This report emphasizes that the condition is a distinctive clinical entity and entirely different from dermatitis herpetiformis and benign chronic bullous dermatosis of childhood.


Thiopyronine-sensitized photodynamic inactivation of RNA synthesis in vitro. The photodynamic inactivation of a cell-free transcriptional system with yeast components was investigated in vitro. The photodynamic inhibition of the overall synthesis is dependent on the irradiation  time and is due to the photo-oxidation of the DNA as well as of the RNA polymerase. Photodynamic treatment of the polymerase and DNA together leads to a stronger inactivation of their biological activity than photodynamic treatment of both components separately. Also the rate of RNA synthesis is more affected when the complete system is photodynamically treated shortly after beginning than before beginning of RNA synthesis. It is concluded that the polymerase-DNA complex is more sensitive to photo-oxidation than the individual components. Because the steps binding and initiation do not seem very affected photodynamically it is suggested that the Thiopyronine-sensitized inhibition of RNA synthesis is mainly due to an inhibition of elongation.


Functional significance of low serum vitamin B12 levels in pregnancy. The physiological significance of low levels of vitamin B12 in serum in late pregnancy was assessed by a functional test viz. urinary excretion of methylmalonic acid (MMA) after a valine load. This was normal in all but one subject despite low levels of vitamin B12 in serum in many of these subjects. Assay of serum vitamin B12 levels in pregnant women and control subjects by two different techniques viz. radio isotope dilution and microbiological methods gave essentially similar results for both groups, although the absolute values in both the groups were over-estimated by the former technique. The data presented indicate that there is no evidence of functional inadequacy in pregnant women with low levels of vitamin B12 in serum.


[Subcorneal pustular dermatosis].

[Subcorneal pustular dermatosis]. Subcorneal dermatosis is a chronic relapsing pustular eruption which has been recognised for 20 years. The diagnosis can be made only by combining the clinical features of a recurrent eruption mainly on the trunk which spares the mucosae and has the histological appearance of a subcorneal bullae filled with polymorphonuclear leucocytes situated on the surface of normal epidermis. No immunofluorescence either direct or indirect similar to that seen in pemphigus, pemphigoid or dermatitis herpetiformis has been reported. No jejunal abnormality has been found in any case but some, though not all, patients respond to treatment with Dapsone (DDS; diaminodiphenylsulfone). The condition can be distinguished from other cases of subcorneal pustular eruptions only by combining the clinical and histological features. The etiology remains unknown.


[Subcutaneous pedicled flap for the reconstruction of the skin mantle following excision of facial tumors]. A subcutaneous pedicled island flap for closing skin defects in face tumor surgery is presented. Simple design and operative technique of these flaps make them interesting for closing excisional face skin defects especially in the elderly.


Biochemical study of minosaminomycin in relation to the kasugamycin group antibiotics. Minosaminomycin is structurally related to kasugamycin and inhibits protein synthesis in mycobacteria. It also inhibits phage f2 RNA-directed protein synthesis in a cell-free system of Escherichia coli by 50% at 2 x 10(-7) M. It is 100-times more potent than kasugamycin in this system. At 10(-7) M minosaminomycin inhibits EF-T dependent binding of aminoacyl-tRNA to ribosomes by 50%. This effect is markedly diminished if minosaminomycin is added to the assay system after a brief incubation of ribosomes with mRNA. Like kasugamycin, minosaminomycin preferentially inhibits the initiation of protein synthesis directed by phage f2 RNA in vitro and does not cause miscoding. Ribosomes from kasugamycin-resistant mutants Ksg A and Ksg C were as sensitive to minosaminomycin as those from each parent strain. In spite of the strong inhibitory activity of minosaminomycin manifested in cell-free systems of E. coli, this compound inhibits the growth of the organism itself only slightly. This discrepancy could be ascribed to impermeability, as E. coli cells with modified permeability show greater sensitivity to minosaminomycin. There is no indication that the antibiotic is activated in E. coli cells. On the basis of these results, the structural features of these antibiotics is inactivated in E. coli cells. On the basis of these results, the structural features of these antibiotics essential for interaction with ribosomes and for permeability into the cells are discussed.


The relative efficacy of methylphenidate (ritalin) and behavior-modification techniques in the treatment of a hyperactive child. Drug versus placebo effects were contrasted with those of contingency management in the treatment of a hyperactive child. Several criterion behaviors were monitored in two different settings to gauge the breadth and generalizability of drug and behavior-management effects. Medication and contingency management effects were both found to be situation specific. No interaction effects were found. Accuracy of task performance, amount of eye contact with the experimenters, frequency of repetitive hand movements, and distractible behavior were apparently unaffected by medication (Ritalin versus placebo) within the clinic. A multiple-baseline design incorporating contingency reversals revealed the reinforcement contingencies to be the crucial variable controlling behavior within the clinic. Medication effects were shown to be significant within the home setting where reinforcement contingencies were not changed. While aggressive behavior decreased as a function of Ritalin, repetitive hand movements increased.


Pyruvate formation during the catabolism of simple hexose sugars by Escherichia coli: studies with pyruvate kinase-negative mutants. Escherichia coli K-12 mutants lacking the adenosine 5'-monophosphate-activated pyruvate kinase have been isolated accidentally and used to prepare further mutants additionally devoid of the fructose bisphosphate-activated pyruvate kinase. Such double mutants totally devoid of pyruvate kinase activity still grow well under aerobic conditions on sugars that are catabolized by the phosphoenolpyruvate (PEP):sugar phosphotransferase system, but they grow poorly on non-phosphotransferase system sugars. This suggests that although pyruvate kinase plays a major role in the formation of pyruvate from PEP during growth on non-phosphotransferase system sugars, the operation of the PEP:sugar phosphotransferase system can contribute significantly to pyruvate production from PEP. In the absence of pyruvate kinase and an active PEP:sugar phosphotransferase system the methylglyoxal glycolytic bypass may also function to some extent for the formation of pyruvate during the catabolism of simple hexose sugars. No unique physiological role can yet be ascribed to the adenosine 5'-monophosphate-activated pyruvate kinase as a result of these studies.


Two unlinked genes for the pyruvate dehydrogenase complex in Aspergillus nidulans. The activity of the overall pyruvate dehydrogenase complex was found to be similar in extracts of Aspergillus nidulans after growth on either sucrose or acetate. Eight mutants lacking the activity of this complex were found among some 200 glycolytic mutants selected for their inability to grow on sucrose. The absence of pyruvate dehydrogenase complex activity was also confirmed for a mutant, g6 (pdhA1), isolated previously. Studies with the mutants supported the existence of two unlinked genes, pdhA and pdhB, controlling the function of the complex. In vivo and in vitro complementation between mutations at the two loci were shown by the ability of forced heterokaryons to grow on sucrose and by the restoration of overall pyruvate dehydrogenase complex activity in mixed cell-free extracts. The mutations were recessive to their wild-type alleles, and the pdhA and pdhB loci were assigned to linkage groups I and V, respectively.


Oscillations in the synthesis of cell wall components in synchronized cultures of Escherichia coli. The rate of cell wall synthesis with respect to both proteins and lipids was determined in synchronized cultures of Escherichia coli B/r. Whereas the rate of total protein synthesis showed an exponential increase with cell age, the rate of incorporation of proteins and lipids into cell wall had a maximum at a cell age of 30 to 35 min, 15 min before cell division. This oscillation was observed in both the cytoplasmic membrane and in the outer membrane of the cell envelope.


Activity of murein hydrolases in synchronized cultures of Escherichia coli. Murein hydrolase activities were analyzed in synchronized cultures of Escherichia coli B/r. Cell wall-bound murein hydrolase activities, including the penicillin-sensitive endopeptidase, increased discontinuously during the cell cycle and showed maximum activity at a cell age of 30 to 35 min (generation time, 43 min). Maximum activity was observed at the same time that the rate of cell wall synthesis reached its maximum. These oscillations depended on the termination of replication: no increase in hydrolase activity was found if deoxyribonucleic acid synthesis was inhibited at an early time in the life cycle. In contrast, the activity of another murein hydrolase that was not tightly bound to the membrane (transglycosylase) increased exponentially with time, even when deoxyribonucleic acid synthesis was inhibited.


Effect of cessation of phospholipid synthesis on the synthesis of a specific membrane-associated bacteriophage protein in Escherichia coli. The major coat protein of the bacteriophage f1 is synthesized during infection of Escherichia coli and becomes tightly associated with the host membrane. This synthesis was studied in conjunction with the strain BB26-36, a mutant defective in phospholipid synthesis, to investigate basic questions concerning membrane protein and phospholipid synthesis. Coat protein synthesis is decreased in the absence of net phospholipid synthesis. The coat protein produced under these conditions is still found tightly associated with the membrane. Resumption of phospholipid synthesis leads to an increase in the synthesis and accumulation of the coat protein. Therefore, a correlation between coat protein and phospholipid synthesis seems to exist. However, the packaging of phage deoxyribonucleic acid into phage particles proceeds in the absence of phospholipid synthesis, and the number of phage particles produced appears to depend only on the amount of coat protein in the membrane.


Role of transport systems in amino acid metabolism: leucine toxicity and the branched-chain amino acid transport systems. The livR locus, which leads to a trans-recessive derepression of branched-chain amino acid transport and periplasmic branched-chain amino acid-binding proteins, is responsible for greatly increased sensitivity toward growth inhibition by leucine, valine, and serine and, as shown previously, for increased sensitivity toward toxicity by branched-chain amino acid analogues, such as 4-azaleucine or 5',5',5'-trifluoroleucine. These phenotypes are similar to those of relA mutants; however, the livR mutants retain the stringent response of ribonucleic acid synthesis. However, an increase in the rate of transport or in the steady-state intracellular level of amino acids in the livR strain cannot completely account for this sensitivity. The ability of the LIV-I transport system to carry out exchange of pool amino acids for extracellular leucine is a major factor in leucine sensitivity. The previous finding that inhibition of threonine deaminase by leucine contributes to growth inhibition is confirmed by simulating the in vivo conditions using a toluene-treated cell preparation with added amino acids at levels corresponding to the internal pool. The relationship between transport systems and corresponding biosynthetic pathways is discussed and the general principle of a coordination in the regulation of transport and biosynthetic pathways is forwarded. The finding that the LIV-I transport system functions well for amino acid exchange in contrast to the LIV-II system provides another feature that distinguishes these systems in addition to previously described differences in regulation and energetics.


Fine-structure map of the histidine transport genes in Salmonella typhimurium. Afine-structure genetic map of the histidine transport region of the Salmonella typhimurium chromosome was constructed. Twenty-five deletion mutants were isolated and used for dividing the hisJ and hisP genes into 8 and 13 regions respectively. A total of 308 mutations, spontaneous and mutagen induced, have been placed in these regions by deletion mapping. The histidine transport operon is presumed to be constituted of genes dhuA, hisJ, and hisP, and the regulation of the hosP and hisJ genes by dhuA  is discussed. The orientation of this operon relative to purF has been established by three-point crosses as being: purF duhA hisJ hisP.


Role of ribosomal protein S12 in peptide chain elongation: analysis of pleiotropic, streptomycin-resistant mutants of Escherichia coli. Some of the spontaneous streptomycin-resistant mutants of Escherichia coli strain C600 exhibit pleiotropic effects in addition to the antibiotic resistance. These effects include decreased growth rates, reduced levels of certain enzymes, and poor support of bacteriophage growth. One of these mutants, strain SM3, was studied further. We have examined the question of whether the reduced growth rate of the mutant SM3 is related to the reduction in relative amounts of ribosomes or to the reduction in the efficiency of ribosomes in protein synthesis. Measurements of alpha, the differential synthesis rate of ribosomal protein, revealed that the protein synthesis effeciency of ribosomes from the mutant strain SM3  was reduced about twofold relative to that of the parent strain C600. Measurements of the induction lag for beta-galactosidase and of the synthesis time of several different molecular-weight classes of proteins indicated that the mutation resulted in a marked reduction in the peptide chain growth rate. This reduction in the chain growth rate probably accounted for most of the observed reduction in the growth rate of the mutant strain. These experimental results show that the strA gene product, the S12 protein of the 30S subunit, is involved in some aspect of protein chain elongation. Presumably this involvement occurs during the messenger ribonucleic acid-directed binding of transfer ribonucleic acid to the ribosome.


Two-carbon assimilative capacity and the induction of isocitrate lyase in Saccharomyces cerevisiae. The yeast Saccharomyces cerevisiae was grown on 10% glucose medium and subsequently transferred to fresh medium containing 2- and 3-carbon substrates. Under these conditions, the yeast rapidly acquired an oxidative capacity, as evidenced by oxygen uptake rates and 14CO2 evolution rates during respiration on ethanol or (14C)acetate. The assimilative capacity for 2-carbon substrates developed more slowly and followed the induction of isocitrate lyase. Washed yeast transferred to the basic medium containing no added carbon substrate possessed only low levels of isocitrate lyase after a 6-h adaptation. After 6 h, isocitrate lyase was present at high levels in cells transferred to a range of ethanol concentrations but was present in only low amounts in cells transferred to acetate. The role of ethanol as an inducer of isocitrate lyase is discussed.


Synthesis of "group polysaccharide" by membranes from Streptococcus pyogenes and its stabilized L-form. Rhamnose and N-acetylglucosamine (GlcNAc) are incorporated from thymidine 5'-diphosphorhamnose and uridine 5-diphospho-N-acetylglucosamine (UDPGlcNAc) into membrane fragments prepared from Streptococcus pyogenes but not into membrane fragements prepared from a stabilized L-form of this organism. Incorporation from TDPrhamnose is partially dependent upon UDPGlcNAc and vice versa. The oligomeric GlcNAc and rhamonose-containing products are easily extracted from membrane particles by sedimentation through detergent solutions. They are substantially extracted into methanol but not into chloroform-methanol (2:1). When product containing both radioactive rhamnose and GlcNAc is deacetylated and hydrolyzed briefy in acid, glucosaminyl rhamnose is obtained, byt not higher oligomers, suggesting that oligomer synthesis in vitro is terminated because unidentified wnzymatic requirements are not satisfied. The data are consistent with the assembly of group A-specific polysaccharide at the cellular membrane with participation of a lipoid anchor (acceptor) molecule.


Deoxyribonucleic acid repair in vitro by extracts of Escherichia coli. Deoxyribonucleic acid (DNA) from bacteriophage T7 has been used to monitor the capacity of gently lysed extracts of Escherichia coli to perform repair resynthesis after ultraviolet (UV) irradiation. Purified DNA damaged by up to 100 J of UV radiation per m2 was treated with an endonuclease from Micrococcus luteus that introduces single-strand breaks in irradiated DNA. This DNA was then used as a substrate to study repair resynthesis by extracts of E. coli. It was found that incubation with the extract and exogenous nucleoside triphosphates under suitable assay conditions resulted in removal of all pyrimidine dimers and restoration of the substrate DNA to its original molecular weight. Repair resynthesis, detected as nonconservative, UV-stimulated DNA synthesis, was directly proportional tothe number of pyrimidine dimers introduced by radiation. The repair mode described here appears to require DNA polymerase I since it does no occur at the restrictive temperature in polA12 mutants, which contain a thermolabile polymerase. The addition of purified DNA polymerase I to extracts made from a polA mutant restores the ability to complete repair at the restrictive temperature.


Expression of cryptopleurine resistance in Saccharomyces cerevisiae. An examination of gene expression in diploids may not always be sufficient for determination of the dominant or recessive character of an allele. In Saccharomyces cerevisiae resistance to cryptopleurine has been attributed to a single recessive nuclear gene, cryl, located on chromosome III. We found, contrary to expectations, that resistance to cryptopleurine is not expressed in diploids that are monosomic for chromosome III. Examination of strains of different ploidy on gradient plates shows that the presence of the sensitive allele in a cell does not affect the level of resistance, but rather the level of resistance is directly related to the ratio of resistant alleles to the number of chromosome sets.


Conservation of transfer ribonucleic acid and 5S ribonucleic acid cistrons in Enterobacteriaceae. The genes for tranfer ribonucleic acid (tDNA) and 5S ribonucleic acid (5SDNA) were isolated from the total deoxyribonucleic acid (DNA) of Escherichia coli. The relatedness of tDNA and 5S from E. coli and other species of Enterobacteriaceae was determined by reassociation of the isolated genes labeled with 32PO4 to unlabeled, unfractionated DNA. Double-stranded DNA was separated from unreacted DNA by hydroxyapatite chromatography. Thermal elution profiles were done to determine the amount of unpaired bases present in related DNA sequences. Relative to total DNA, both 5S DNA and tDNA were highly conserved throughout the Enterobacteriaceae, including the genera Yersinia and Proteus.


Temporal sequence of events during the initiation process in Escherichia coli deoxyribonucleic acid replication: roles of the dnaA and dnaC gene products and ribonucleic acid polymerase. Three thermosensitive deoxyribonucleic acid (DNA) initiation mutants of Escherichia coli exposed to the restrictive temperature for one to two generations were examined for the ability to reinitiate DNA replication after returning to the permissive temperature in the presence of rifampin, chloramphenicol, or nalidixic acid. Reinitiation in the dnaA mutant was inhibited by rifampin but not by chloramphenicol, whereas renitiation was not inhibited by rifampin but not by chloramphenicol, whereas reinitiation was not inhibited in two dnaC mutants by either rifampin or chloramphenicol. To observe the rifampin inhibition, the antibiotic must be added at least 10 min before return to the permissive temperature. The rifampin inhibition of reinitiation was not observed when a rifampin-resistant ribonucleic acid ((RNA) polymerase gene was introduced into the dnaA mutant, demonstrating that RNA polymerase synthesizes one or more RNA species required for the initation of DNA replication (origin-RNA). Reinitiation at 30 degrees C was not inhibited by streptolydigin in a stretolydigin-sensitive dnaA muntant. Incubation in the presence of nalidixic acid prevented subsequent reinitiation in the dnaC28 mutant but did not inhibit reinitiation in the dnaA5 muntant. These results demonstrate that the dnaA gene product acts before or during the synthesis of an origin-RNA, RNA polymerase synthesizes this origin RNA, and the dnaC gene product is involved in a step after this RNA synthesis event. Furthermore, these results suggest that the dnaC gene product is involved in the first deoxyribounucleotide polymerization event wheareas the dnaA gene product acts prior to this event. A model is presented describing the temporal sequence of events that occur during initiation of a round of DNA replication, based on results in this and the accompanying paper.


NOVEL Escherichia coli dnaB mutant: direct involvement of the dnaB252 gene product in the synthesis of an origin-ribonucleic acid species during initiaion of a round of deoxyribonucleic acid replication. The initiation process of deoxyribonucleic acid (DNA) replication in Escherichia coli has been studied using the thermoreversible dna initiation mutant E. coli HfrHl65/120/6 dna-252. This dna mutation was incorrectly classed as a dnaA mutation. Biochemical and genetic evidence suggests that the dna-252 mutant is a novel dnaB mutant, possessing phenotypic properties which distinguish it from other dnaB mutants. Sensitivity of reinitiation in the dna-252 mutant to specific inhibitors of protein, ribonucleic acid (RNA), and DNA synthesis was studied. Reinitiation is shown to be sensitive to rifampin and streptolydigin but not to cholramphenicol. Thus, the dna-252 gene product appears to be required during the initiation process for a step occurring either before or during synthesis of an RNA species (origin-RNA). Using reversible inhibition of RNA synthesis by streptolydigin of a streptolydigin-sensitive derivative of the dna-252 mutant, the dna-252 gene product is shown to be directly involved in the synthesis of an orgin-RNA species. These results are included in a schematic model presented in the accompanying paper of the temporal sequence of events occurring during the initiation process.


Growth of the Escherichia coli cell surface. Phage T6 was used as a label to follow the growth of the outer membrane in a strain of Escherichia coli temperature sensitive for the production of the T6 receptor. Extension of the surface takes place at the cell poles. Small cells extend at only one pole, whereas larger cells grow from both poles. The change from unipolar to bipolar growth appears to depend on the attainment of a particular cell size and not on completion of chromosome replication.


Effects of lipid phase transition of the freeze-cleaved envelope of Escherichia coli. We studied the fine structure of the envelope of Escherichia coli auxotroph K1060 after the cells were grown in the presence of one of the following fatty acids; oleic, palmitelaidic, or elidic acid. The cells were freeze-fractured after exposure to temperatures above and below the lipid phase transition range. As judged by freeze-etching methods, we observed that below the transition range the fracture plane of the inner membrane showed the typical aggregation of intramembranous particles (IMP) and concomitant development of areas devoid of IMP. In these areas we found a regular arrangement of equally spaced ridges, often intersected at 90 degrees by arrays of similar ridges. The ridges were composed of spherical particles measuring 4 to 5 nm in  diameter. Formation and melting of these arrays took place within 15 to 30s after temperature shift-down or shift-up, respectively. Fixation in glutaraldehyde prevented these changes. The outer-membrane fracture plane revealed ordered areas to a lesser degree; these were discernible only by the regular arrangement of the IMP of the concave fracture plane. We interpret the data by suggesting that the pattern of ridges in E. coli K1060 is analogous to the band patterns described for artificial liposomes, and that the particles, possibly  proteins, are lined up or extruded along the ridges during membrane lipid crystallization.