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Influenza and other respiratory viruses present a significant threat to public health, national security, and the world economy, and can lead to the emergence of global pandemics such as from COVID-19. A barrier to the development of effective therapeutics is the absence of a robust and predictive preclinical model, with most studies relying on a combination of in vitro screening with immortalized cell lines and low-throughput animal models. Here, we integrate human primary airway epithelial cells into a custom-engineered 96-device platform (PREDICT96-ALI) in which tissues are cultured in an array of microchannel-based culture chambers at an air–liquid interface, in a configuration compatible with high resolution in-situ imaging and real-time sensing. We apply this platform to influenza A virus and coronavirus infections, evaluating viral infection kinetics and antiviral agent dosing across multiple strains and donor populations of human primary cells. Human coronaviruses HCoV-NL63 and SARS-CoV-2 enter host cells via ACE2 and utilize the protease TMPRSS2 for spike protein priming, and we confirm their expression, demonstrate infection across a range of multiplicities of infection, and evaluate the efficacy of camostat mesylate, a known inhibitor of HCoV-NL63 infection. This new capability can be used to address a major gap in the rapid assessment of therapeutic efficacy of small molecules and antiviral agents against influenza and other respiratory viruses including coronaviruses.

In the Nursing Standard supplement on catheter care, which I read and found very interesting, I came across a point which I intended to write about at the time but have been side tracked by other things.

Calcitonin gene-related peptide (CGRP) was examined for its effects on intracellular free Ca2+ concentration ([Ca2+]i) in UMR 106 osteoblast-like cells. Cells loaded with the Ca2+ dye FURA-2 dose-dependently responded to CGRP (1-100 nM) with transient two-fold increases in [Ca2+]i. An intracellular source for this Ca2+ transient was suggested by the failure of membrane depolarization with high extracellular K+ or acute depletion of extracellular Ca2+ ([Ca2+]e) with EGTA to attenuate this response. After cells were incubated for 45 min with 0.1 mM extracellular Ca2+ to deplete intracellular Ca2+ stores, CGRP produced a 25-30% decrease in [Ca2+]i rather than a transient increase. This calcium decrease was mimicked by membrane depolarization or by pinacidil, a specific activator of adenosine triphosphate (ATP)-sensitive potassium (KATP) channels, and blocked by glybenclamide, a specific blocker of KATP channels. Our data suggest that CGRP has diverse Ca2+ regulatory effects in UMR 106 cells, mobilizing Ca2+ from intracellular stores via classical signaling while possibly promoting cellular Ca2+ efflux or inhibiting uptake through voltage-dependent Ca2+ channels via KATP-mediated hyperpolarization.

During evolution of fungi, the separate tryptophan synthetase alpha and beta polypeptides of bacteria appear to have been fused in the order alpha-beta rather than the beta-alpha order that would be predicted from the order of the corresponding structural genes in all bacteria. We have fused the tryptophan synthetase polypeptides of Escherichia coli in both orders, alpha-beta and beta-alpha, with and without a short connecting (con) sequence, to explore possible explanations for the domain arrangement in fungi. We find that proteins composed of any of the four fused polypeptides, beta-alpha, beta-con-alpha, alpha-beta, and alpha-con-beta, are highly active enzymatically. However, only the alpha-beta and alpha-con-beta proteins are as active as the wild type enzyme. All four fusion proteins appear to be less soluble in vivo than the wild type enzyme; this abnormal characteristic is minimal for the alpha-con-beta enzyme. The alpha and beta domains of the four fusion polypeptides were not appreciably more heat labile than the wild type polypeptides. Competition experiments with mutant tryptophan synthetase alpha protein, and the fusion proteins suggest that in each fusion protein the joined alpha and beta domains have a functional tunnel connecting their alpha and beta active sites. Three tryptophan synthetase beta'-alpha fusion proteins were examined in which the carboxyl-terminal segment of the wild type beta polypeptide was deleted and replaced by a shorter, unnatural sequence. The resulting deletion fusion proteins were enzymatically inactive and were found predominantly in the cell debris. Evaluation of our findings in relation to the three-dimensional structure of the tryptophan synthetase enzyme complex of Salmonella typhimurium (5) and the results of mutational analyses with E. coli suggest that tryptophan synthetase may have evolved via an alpha-beta rather than a beta-alpha fusion because in beta-alpha fusions the amino-terminal helix of the alpha chain cannot assume the conformation required for optimal enzymatic activity.

Dramatic changes in smoking behaviour occurred over the half century since cigarette smoking was identified as a cause of human disease, and these changes are a major public health accomplishment.1 Perhaps no component of this change has been more dramatic than the reduction in people’s involuntary exposure to tobacco smoke pollution. The fraction of indoor workers protected by a total ban on smoking in the workplace has risen from 3% in 19862 to nearly 70% in 1999,3 and serum cotinine levels in non-smokers declined 70% between 1988 and 1998.4 Much of this progress can be attributed to the courageous and determined actions of individuals applying our advances in the medical sciences. This issue both celebrates those individuals and details their accomplishments. Almost immediately following Surgeon General Jesse Steinfeld’s articulation of involuntary smoking as a public health issue and his declaration of a non-smokers bill of rights,5 airlines became the leading edge of a societal change that would de-normalise smoking and protect non-smokers from exposure to tobacco smoke. As presented by Holm and Davis6 in this issue, aircraft were one of the first public places to have separate smoking and non-smoking sections, and regulation of airline travel would continue to lead the rest of society toward banning smoking in the work environment for the next two decades. Efforts to regulate exposure to tobacco smoke in air travel culminated in the elimination of all smoking on domestic flights lasting less than two hours in 1988, a total ban on domestic flights in 1990, and a ban on international flights in 2000. Progress on airplanes was a highly visible …

E. coli UshA, a bifunctional enzyme with UDP-sugar hydrolase and 5'-nucleotidase activities, is secreted to the periplasm but has a specific protein inhibitor located in the cytoplasm. It has been previously suggested that some 5'-nucleotidase, or a folded domain of this enzyme, may be active in the cytoplasm prior to export. If true, the intracellular inhibitor may have a role in protecting the cell from the likely deleterious effects of any intracellular UshA activity. Using deletion mutagenesis to remove the UshA signal peptide, we have shown that the resulting UshA derivative is an active cytoplasmic 5'-nucleotidase, and causes conditional lethality. Our results support the hypothesis that the physiological role of the UshA inhibitor is to protect the intracellular nucleotide pool from any cytoplasmic 5'-nucleotidase activity.

Cigarettes with lower machine measured "tar" and nicotine yields have been marketed as "safer" than high tar products over the last four decades, but there is conflicting evidence about the impact of these products on the disease burden caused by smoking. This paper critically examines the epidemiological evidence relevant to the health consequences of "reduced yield" cigarettes. Some epidemiological studies have found attenuated risk of lung cancer but not other diseases, among people who smoke "reduced yield" cigarettes compared to smokers of unfiltered, high yield products. These studies probably overestimate the magnitude of any association with lung cancer by over adjusting for the number of cigarettes smoked per day (one aspect of compensatory smoking), and by not fully considering other differences between smokers of "high yield" and "low yield" cigarettes. Selected cohort studies in the USA and UK show that lung cancer risk continued to increase among older smokers from the 1950s to the 1980s, despite the widespread adoption of lower yield cigarettes. The change to filter tip products did not prevent a progressive increase in lung cancer risk among male smokers who began smoking during and after the second world war compared to the first world war era smokers. National trends in vital statistics data show declining lung cancer death rates in young adults, especially males, in many countries, but the extent to which this is attributable to "reduced yield" cigarettes remains unclear. No studies have adequately assessed whether health claims used to market "reduced yield" cigarettes delay cessation among smokers who might otherwise quit, or increase initiation among non-smokers. There is no convincing evidence that past changes in cigarette design have resulted in an important health benefit to either smokers or the whole population. Tobacco control policies should not allow changes in cigarette design to subvert or distract from interventions proven to reduce the prevalence, intensity, and duration of smoking. Keywords: tar and nicotine; lung cancer; changing cigarette

Test whether a change in oxygen consumption produced by a reduction in level of mechanical ventilatory support predicts failure to tolerate the reduction in level of support.Prospective study of the sensitivity and specificity of increased oxygen cost of breathing as a predictor of failure to tolerate a reduction in ventilatory support in patients undergoing weaning, using a protocol that incrementally reduces the level of mechanical ventilatory support.University medical center.We studied 228 trials in 30 patients who had required mechanical ventilatory support for at least 72 hours and who were being weaned using a standardized protocol that provided for three 30-minute trials of reduced mechanical ventilatory support per day, followed by ventilatory muscle rest. Using a metabolic monitor, we monitored oxygen consumption (V(O(2))) prior to and during 228 incremental reductions in level of mechanical ventilatory support conducted as part of a standardized weaning protocol. Oxygen cost of breathing was defined as the difference in V(O(2)) (Delta V(O(2))) during the trial of reduced mechanical ventilatory support, compared to a 30-minute resting period immediately before the trial. A successful trial was defined as one that could be continued for 30 minutes without development of clinical signs of ventilatory failure. Changes in V(O(2)) and the ratio of respiratory frequency to tidal volume (f/V(T)) during a weaning trial were evaluated as predictors for failure of a 30-minute trial of reduced ventilatory support.A 15% increase in oxygen cost of breathing predicted failure in the trial, with a sensitivity of 96.6%, specificity of 85.7%, positive predictive value of 98.5%, and negative predictive value of 72.0%. Neither change in V(O(2)) measured early in the trial nor f/V(T) proved to be as successful in predicting failure to tolerate an incremental reduction in ventilatory support.Change in V(O(2)) following an incremental reduction in level of mechanical ventilatory support may be a useful predictor for determining which patients will rapidly fail to tolerate that level of reduction.

Cigarette smoking is the major cause of lung cancer, and most lung cancers have historically occurred among current cigarette smokers or recent quitters. The relative risk of developing lung cancer declines in former smokers to approximately twice that of never smokers after 20 years of cessation, but it remains elevated indefinitely. As an increasing fraction of those who have ever smoked become former smokers, the absolute risk of lung cancer in the population will decline; and an increasing fraction of lung cancer cases will occur among former smokers.Data from the 12-year follow-up of American Cancer Society Cancer Prevention Study I are used to estimate lung cancer death rates for smokers, never smokers, and former smokers by duration of cessation. These estimates are combined with population-based estimates of smoking status by birth cohort to generate models of lung cancer death rates for current and former smokers.Modeled estimates of lung cancer death rates suggest that, in the near future, the majority of lung cancer cases will be occurring among former smokers.Former smokers already have acted on the advice of their physicians to reduce their lung cancer risk; however, they will generate an increasing fraction of the cancers that are seen. Approaches must be developed for these former smokers to alter their risk if lung cancer death rates are going to continue to be affected. Additional primary preventive strategies, including chemoprevention, offer hope to these former smokers, as do secondary preventive efforts, such as screening.

To determine the prevalence of smoking policies in indoor work environments in the United States, with a special focus on sex differences in the provision of these policies.Information on the prevalence and restrictiveness of workplace smoking policies was obtained from 86,490 currently employed indoor workers (50,865 women and 35,625 men) 15 years of age and older who responded to the National Cancer Institute's Tobacco Use Supplement to the Current Population Survey, a cross-sectional survey of households in all 50 states and the District of Columbia conducted between 1995 and 1996.Eighty-six percent of respondents reported that their workplaces had official smoking policies, and 63% reported that their workplaces were smoke free. Women reported significantly higher rates of both official smoking policies and smoke-free workplaces than men, regardless of racial/ethnic or age group.The overall rates of worksite smoking restrictions, including the establishment of smoke-free workplaces, were higher than those reported in earlier surveys. Disparities in coverage will need to be reduced if all workers, regardless of sex, race, age, or industry of employment, are to be protected from the demonstrated hazards of environmental tobacco smoke.

During surgical treatment of periodontal disease, enamel matrix derivative (EMD) is topically applied as a substitute for extracellular matrix in order to facilitate regeneration of damaged periodontal tissue. However, the mechanism for EMD action is poorly understood. We have now examined the effects of EMD on the proliferation of oral epithelial (SCC25) cells in vitro. After 3 days of treatments, EMD (25 100 microg/ml) dose-dependently inhibited cell division and concomitantly arrested cell cycle at the G1 phase. Prior to this inhibition, EMD significantly up-regulated p21WAF1/cip1, a cyclin-dependent kinase inhibitor, induced G1-arrest, and inhibited DNA synthesis. In addition, EMD down-regulated expression of cytokeratin-18 (CK18) protein, which was most due to decreased production, but less to increased degradation. However, EMD did not discernibly increase the number of apoptotic cells over 8 days of treatment. These findings indicate (1) that EMD acts as a cytostatic agent, rather than a cytotoxic agent, on epithelial cells, and (2) that this anti-proliferative action is probably due to p21WAF1/cip1-mediated G1-arrest. Furthermore, our in vitro cellular data clearly verify and provide an explanation for the clinical observation that EMD application suppresses the down-growth of junctional epithelium onto dental root surfaces, a process that frequently interferes with the formation of new connective tissue attachments.

In previous studies, we have shown that calcitonin gene-related peptide (CGRP) acutely inhibits 45Ca2+ uptake in osteoblastic UMR106 cells, and we have proposed that ATP-sensitive potassium (K(ATP)) channels are involved in mediating this action of CGRP. To directly test this proposed mechanism, we have now examined the effects of CGRP on both membrane potential (Em) and K+ mobilization in UMR106 cells, using specific fluorometric dye assays. CGRP (0.01-100 nM) induced membrane hyperpolarization in a dose-dependent manner, with a half maximal effect (ED50) at approximately 0.2 nM and a maximal effect at 100 nM. Both pinacidil (Pina; a K(ATP) channel opener) and forskolin (FSK) induced similar membrane hyperpolarization, but the actions of these three agents could be easily distinguished: both CGRP and Pina actions were well antagonized by glibenclamide (Glib; a selective K(ATP) channel blocker), whereas FSK action was strongly attenuated only by tetraethylammonium (a K(Ca) channel blocker) or compound H-89 (an inhibitor of cAMP-dependent protein kinases). Cells pretreated with Pina no longer responded to CGRP, but they could still respond to FSK; furthermore, pretreatment with FSK failed to block successive treatment with either CGRP or Pina. In parallel with observed changes in Em, CGRP (0.01-100 nM) decreased intracellular K+ concentrations ([K+]i) in a dose-dependent manner, with an ED50 identical to that obtained for alterations in Em. This action of CGRP was sensitive to Glib and had only slight sensitivity to tetraethylammonium; this CGRP effect was mimicked by Pina but not by FSK. Interestingly, CGRP significantly elicited changes in cell shape by a Glib-sensitive mechanism that included notable decreases in cross-sectional cytoplasmic area. These observations strongly support our proposal that CGRP primarily stimulates K+ efflux via activation of K(ATP) channels and thereby induces membrane hyperpolarization in UMR106 cells. Furthermore, our data also suggest that this cascade of initial cellular events may result in rapid changes in cell morphology and decreases in cellular area of the type that are thought to act as triggers for proliferation and/or differentiation in many cellular phenotypes.

Media messages have the potential to change public health. Changes in media have been associated with major changes in smoking behavior, but only when the rest of the social structure actively changed the environment for the smoker. These changes act synergistically with media messages, and cessation or behavior change occurs. Only when we put media into the larger construct of the society within which we live, and when that society is receptive to media messages, can one expect to see an effect. It is perhaps not a specific attitude change that is so critical, but rather the presence of those messages which can grow in the smoker's environment and eventually become strong enough to allow that smoker to quit for the long term.

Escherichia coli contains a single periplasmic UDP-glucose hydrolase (5'-nucleotidase) encoded by ushA. Salmonella enterica, serotype Typhimurium, also contains a single UDP-glucose hydrolase but, in contrast to E. coli, it is membrane-bound and is encoded by the non-homologous ushB gene; Salmonella enterica (Typhimurium) also contains a silent allele of the ushA gene (ushA0). In this report, we show that nearly all natural isolates of Salmonella contain both UDP-sugar hydrolases, i.e. they are UshA+ UshB+. The only exceptions are all from sub-group I (S. gallinarum, S. pullorum, and most Typhimurium strains), are UshA- UshB+, and several have been shown to contain an ushA0 allele. These data, together with the fact that these latter strains are closely related genetically, strongly suggests a recent silencing mutation(s). We also report the presence in E. coli K-12, and in natural isolates of E. coli, of a DNA sequence which is homologous to the ushB gene of Salmonella; since E. coli does not contain UshB activity, we tentatively refer to this sequence as ushB0. Since all E. coli strains investigated are UshB-, we conclude that the silencing mutation(s) occurred relatively early following the divergence of Escherichia coli and Salmonella from a common ancestor that was ushA+ ushB+.

To determine the extent of exposure of nonsmoking indoor workers to environmental tobacco smoke (ETS) according to type of work-site smoking policy, work area, workplace size, and demographic characteristics.Participants included 7162 adult, nonsmoking, indoor workers who were interviewed as part of the 1990 California Tobacco Survey. Respondents were asked whether anyone had smoked in their work area within the past 2 weeks.An estimated 2.2 million California nonsmokers were exposed to tobacco smoke at indoor work sites in 1990. Nonsmoker exposure to ETS was 9.3% for those working in a smoke-free worksite, 23.2% for those working where there was only a work-area restriction, 46.7% for those working where there was a policy that did not include the work area, and 51.4% for those working where there was no work-site smoking policy. After adjustment for type of work area (eg, office, open area), workplace size, and demographic factors, it was determined that nonsmokers working where there was only a work-area ban were 2.8 times more likely to be exposed to ETS than those working in a smoke-free work site. In workplaces with no policy or a policy not covering the work area, nonsmokers were over eight times more likely to be exposed to ETS than those who worked in a smoke-free work site. Nonsmokers who were 18 to 24 years of age, male, or Hispanic, and those with less than a high school education had more exposure to ETS.These results indicate that adequate protection of nonsmokers from ETS exposure requires a smoke-free work site.

Various smoking intervention approaches have been demonstrated to successfully alter smoking behavior among individual smokers, but it is difficult to demonstrate the benefit of these individual cessation approaches across the population of smokers. In contrast, efforts that concentrate on altering the social and economic environment within which the smoker smokes, most notably the media, taxation, and changing the social acceptability of smoking, have been linked to substantial shifts in the smoking behavior of the US population. Attacking tobacco use as a form of sociological carcinogenesis, rather than focusing on the individual smoker, allows alteration at the root of smoking behavior, ie, its personal, social, and psychological utility for the smoker.

Tobacco use was widespread in the New World by the time of the first voyage of Columbus; however, it is only in the last century that the use of tobacco as cigarettes has been prevalent. The milder tobacco and more acidic smoke of cigarettes lead to the deeper inhalation of tobacco into the lung with resultant deposition and absorption of the addicting, toxic, and carcinogenic components of the smoke. More than 4000 individual constituents have been identified in cigarette smoke, and the relative concentrations of these constituents vary widely between brands of cigarettes. Tar yield, a measure of the total particulate matter of the smoke, varies markedly with the characteristics of the cigarette manufacture and with the pattern of inhalation. As a result, tar is not a good measure of the dose of toxic or carcinogenic agents received by the individual smoker. The particle size of cigarette smoke is in the range that will lead to deposition in the airways and alveoli of the lung, and many of the gas-phase constituents are absorbed across the alveolar capillary membrane. The irritant agents in the smoke cause acute and chronic changes in lung structure and function that may result in greater retention of carcinogens within the lung and increased vulnerability of the lung to the effects of these carcinogens. Carcinogens and other constituents of cigarette smoke are also absorbed into the blood and metabolized to active forms through microsomal enzyme systems induced by cigarette smoke. The cellular influx of neutrophils and alveolar macrophages that is part of the inflammatory response may be the precursor of the alveolar wall destruction that results in emphysema. The prevalence of smoking is not uniformly distributed across the population. Men began smoking in large numbers very early in the century, but women began to smoke in large number only at the time of the Second World War. Men born after 1930 have been less likely to take up smoking than their older counterparts. The prevalence of smoking is currently declining in both men and women.

None

The cpdB gene encodes a periplasmic 2',3'-cyclic phosphodiesterase (3'-nucleotidase). This enzyme has been purified previously and the gene is located at 96 min on the Escherichia coli chromosome. In this study the cpdB gene was cloned from ClaI-cleaved DNA, and the gene product was identified. DNA blotting experiments showed that the recombinant plasmid contains a deletion with respect to the expected genomic fragment of approximately 4 kilobases, which extends into the vector. Furthermore, the gene was absent from three other recombinant libraries. Together, these findings suggest the presence in the genome of an adjacent gene whose product is lethal when it is present on a multicopy plasmid. The nucleotide sequence of the cpdB gene was also determined. The 5' and 3' untranslated sequences contain characteristic sequences that are involved in the initiation and termination of transcription, including two possible promoters, one of which may contain two overlapping -10 sequences. A strong Shine-Dalgarno sequence is followed by an open reading frame which corresponds to a protein having a molecular weight of 70,954. The first 19 amino acid residues have the characteristics of a signal peptide. The 3' untranslated sequence contains two putative rho-independent transcription terminators having low thermodynamic stability.

Adult, male rats were maintained under 12L:12D with lights on at 06.00h. Their pineal glands were incubated at 37 degrees C in the presence or absence of 10(-4)M norepinephrine (NE). 5-HT and various metabolites were quantitated in post-incubation media and pineal glands by high performance liquid chromatography coupled with electrochemical detection. No differences were observed in the quantities of 5-HT released by pineal glands in four hour incubations starting at either 06.00, 13.00 or 18.00 h; however, a highly significant decrease below these levels was observed at 01.00h. NE significantly stimulated 5-HT release at 13.00 and 18.00 h, but was ineffective at 01.00 and 06.00h. These results confirm recently reported stimulatory effects of NE on the release of 5-HT into pineal gland incubation medium and further suggest a diurnal rhythm of pineal gland sensitivity to NE in vitro with maximum stimulation of 5-HT release at midphotophase.

Rat liver glucosidase II, an endoplasmic reticulum hydrolase involved in the biosynthesis of the N-linked class of glycoproteins, has been purified in good yield to a state approaching homogeneity. The purified enzyme hydrolyzes p-nitrophenyl-alpha-D-glucopyranoside, 4-methylumbelliferyl-alpha-D-glucopyranoside, maltose, and the precursor oligosaccharides glucose1-2mannose9N-acetylglucosamine, but it does not act on glucose3mannose9N-acetylglucosamine or p-nitrophenyl-beta-D-glucopyranoside. The ratio of the rate at which glucose is released from p-nitrophenyl-alpha-D-glucopyranoside to that from glucose2mannose9N-acetylglucosamine or glucose1mannose9N-acetylglucosamine remains constant throughout the 8-step purification procedure; thus it appears that a single enzyme is responsible for the activities toward both the artificial and oligosaccharide substrates. The fact that the enzyme cleaves both of the inner 1,3-linked glucosyl residues from the precursor oligosaccharides supports the view that they are linked in the alpha-configuration. The pH dependence of enzymatic activity is quite similar for different substrates, showing a broad optimum between pH 6 and 7.5. Activity toward p-nitrophenyl-alpha-D-glucopyranoside is enhanced by 12 mM 2-deoxy-D-glucose (260-300% activation) and 25 mM mannose (150% activation), but these two compounds inhibit the action of the enzyme toward the precursor oligosaccharides. By isoelectrofocusing the purified enzyme exhibits one form, which has a pI of 3.5-3.8. Reductive polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicates that glucosidase II has a subunit molecular weight of 65,000. Ferguson plot analysis of the behavior of native enzyme in polyacrylamide gels indicates that it is a 262,000-dalton tetramer. Gel filtration gives a molecular weight of 288,000. Several lines of evidence indicate that the enzyme is a glycoprotein.

By testing serum samples taken between 1980 and 1984 from men attending a department of sexually transmitted diseases, it was shown that antibodies to human immunodeficiency virus (HIV) first appeared in 1981. Homosexual men were significantly more likely to have antibodies to HIV and to cytomegalovirus (CMV) than were heterosexual men attending the same clinic. This shows that homosexuals are exposed to both HIV, the cause of the acquired immune deficiency syndrome (AIDS), and to CMV, which can reactivate to cause life threatening disease once immunosuppression has developed. All homosexuals, not just those with antibodies against HIV, had raised levels of CMV antibodies. This suggests that they experience frequent antigenic stimulation after reinfections with CMV or reactivation of endogenous virus.

In [23], Schiffer and Spencer prove that all small deformations of complex structure on a compact Riemann surface may be realized by altering the complex structure only within an arbitrarily small neighborhood of a point on the surface. It seems interesting in general to consider whether it is possible to construct deformations of an algebraic variety or complex manifold from deformations of neighborhoods of certain subvarieties. Further motivation for trying to understand the role of subvarieties in deformations is suggested by the instability under deformation of the Neron-Severi group, i.e., the group of divisors modulo numerical equivalence. As an example, one may consider the family of affine surfaces Vt: x 2 + y 2 + z 2 = t z (t is a parameter; V o has a nodal singularity at the origin). This family admits a resolution {Xt} --, {V~}, with {X,} a smooth family of non-singular surfaces, and each X~ is a minimal resolution of V t ([4] or [5]). The exceptional curve E in X o is a IP 1 with self-intersection 2 which does not appear in any Xt, for t#:0. One may ask whether every smooth surface X with such a curve in it admits a one-parameter family of deformations arising from this local model. Furthermore, if X contains several disjoint such curves, does each one independently contribute one dimension to the moduli of X? The Hartogs ' theorem of [14] says that one cannot simply plumb in the local deformation, leaving the structure of X unchanged outside a small neighborhood of E. Moreover, old examples of Segre [26] show that the nodes on certain hypersurfaces V in IP 3 are not "independent", i.e., there aren't enough deformations of the resolution X of V to allow for a one-dimensional contribution from each node. Theorem (3.7) of this paper says that the regularity of the Kuranishi variety of X is sufficient for the deformations of X to realize independently

As a first step in studying the biosynthesis of the peptide hormone calcitonin, we have identified procalcitonin species in CA-77 cells, a newly developed rat medullary thyroid carcinoma cell line. mRNA extracted from the cells directed the synthesis of a putative procalcitonin in a reticulocyte lysate translation system containing microsomal membranes. Both this species and a radiolabeled form of immunoreactive calcitonin from intact cells had the same retention time during reverse phase high performance liquid chromatography. The putative cellular procalcitonin was also immunoprecipitated by antiserum to a synthetic peptide whose sequence constitutes the COOH-terminal 16 residues of preprocalcitonin. The polypeptide had a Mr = 13,400, as estimated by gel filtration chromatography under denaturing conditions. Microsequencing of the [35S]methionine-labeled polypeptide indicated that residues 13, 32, and 34 of procalcitonin were methionine. Similar analysis of the peptide labeled with [3H]proline indicated that residues 2 and 11 of the precursor were proline. The positions of methionine and proline could be aligned in a unique manner with the NH2-terminal half of the preprocalcitonin sequence inferred from cDNA analyses. These results indicate that procalcitonin consists of 111 amino acids and suggest that a 25-residue signal sequence is cotranslationally cleaved from preprocalcitonin. From the procalcitonin sequence we can now predict the sequence of likely biosynthetic intermediates and mature secretory products derived from the NH2-terminal as well as COOH-terminal regions of the precursor.

The complete nucleotide sequence of the Neurospora crassa trp-3 gene-encoding tryptophan synthetase has been determined; we present an analysis of its structure. A comparison of the deduced amino acid sequence of the trp-3 polypeptide with its homologs in Saccharomyces cerevisiae (encoded by the TRP5 gene) and Escherichia coli (encoded by the trpA and trpB genes) shows that the A and B domains (amino acid segments homologous to the trpA and trpB polypeptides, respectively) of the N. crassa and yeast polypeptides are in the same order (NH2-A-B-COOH). This arrangement is the reverse of the gene order characteristic of all prokaryotes that have been examined. N. crassa tryptophan synthetase has strong homology to the yeast TRP5 polypeptide (A domains have 54% identity; B domains have 75% identity), and somewhat weaker homology to the E. coli trpA and trpB polypeptides (A domains have 31% identity; B domains have 50% identity). The two domains of the N. crassa polypeptide are linked by a connector of 54-amino acid residues that has less than 25% identity to the 45-residue connector of the yeast polypeptide, although secondary structure analysis predicts both connectors would be alpha-helical. In contrast to the yeast TRP5 gene, which has no introns, the trp-3 coding region is interrupted by two introns 77 and 71 nucleotides in length. Both introns are located near the 5'-end of the gene and therefore not near the segment encoding the connector.

Fluorene, C 13 H 10 , crystallizes in the orthorhombic system, space group Pnam with four molecules per unit cell. The molecule possesses a plane of symmetry which is parallel to the (001) plane of the crystal. The structure has been determined by trial-and-error methods, followed (i) by a least-squares refinement and (ii) by two-dimensional Fourier syntheses. The results from the two methods of refinement are compared and for (ii) a method is given for systematically correcting for overlapping of atoms in projections. The accuracy of the co-ordinates given by these methods is discussed in detail, and it is concluded that the co-ordinates adopted are correct to ±0.017 Å. The carbon skeleton is planar to within the experimental error and the equation of the plane is y = 1.4375 x + 11.0947.

The co-ordinates of the carbon and hydrogen atoms and the anisotropic thermal vibrations of the carbon atoms of chrysene have been determined by three-dimensional least-squares refinement and by three-dimensional Fourier, and difference Fourier, syntheses. The value of R = Ʃ || F o | - | F c ||/Ʃ| F o for over 1000 planes observed with Cu Kα radiation is 0·076. The standard deviations of the carbon atom co-ordinates are less than 0·0033 Å. One C–C bond is significantly longer than the mean and longer than would be expected by any theoretical consideration. The value 1·465 Å can be explained by overcrowding of the hydrogen atoms attached to the carbon atoms concerned. The ‘K’ bond of the phenanthrene nucleus has a length of 1·365 Å and this is in the same benzene ring and diametrically opposite to the very long bond mentioned above. It is shown that the thermal vibrations obtained for the individual carbon atoms can be well represented by considering the molecule to vibrate as a rigid body.

A method of obtaining accurate film-to-film correlation in the study of the diffuse scattering of X-rays by the systematic Laue method is described. It is shown how the symmetry of the crystal leads to a multiplicity of measured calibration ratios, and detailed results from three Laue photographs of hexamine are discussed. A final accuracy of about 1% in the film-to-film ratios is achieved.

As a first step in studying the biosynthesis of the peptide hormone calcitonin, we have identified procalcitonin species in CA-77 cells, a newly developed rat medullary thyroid carcinoma cell line. mRNA extracted from the cells directed the synthesis of a putative procalcitonin in a reticulocyte lysate translation system containing microsomal membranes. Both this species and a radiolabeled form of immunoreactive calcitonin from intact cells had the same retention time during reverse phase high performance liquid chromatography. The putative cellular procalcitonin was also immunoprecipitated by antiserum to a synthetic peptide whose sequence constitutes the COOH-terminal 16 residues of preprocalcitonin. The polypeptide had a Mr = 13,400, as estimated by gel filtration chromatography under denaturing conditions. Microsequencing of the [35S]methionine-labeled polypeptide indicated that residues 13, 32, and 34 of procalcitonin were methionine. Similar analysis of the peptide labeled with [3H]proline indicated that residues 2 and 11 of the precursor were proline. The positions of methionine and proline could be aligned in a unique manner with the NH2-terminal half of the preprocalcitonin sequence inferred from cDNA analyses. These results indicate that procalcitonin consists of 111 amino acids and suggest that a 25-residue signal sequence is cotranslationally cleaved from preprocalcitonin. From the procalcitonin sequence we can now predict the sequence of likely biosynthetic intermediates and mature secretory products derived from the NH2-terminal as well as COOH-terminal regions of the precursor.

The complete nucleotide sequence of the Neurospora crassa trp-3 gene-encoding tryptophan synthetase has been determined; we present an analysis of its structure. A comparison of the deduced amino acid sequence of the trp-3 polypeptide with its homologs in Saccharomyces cerevisiae (encoded by the TRP5 gene) and Escherichia coli (encoded by the trpA and trpB genes) shows that the A and B domains (amino acid segments homologous to the trpA and trpB polypeptides, respectively) of the N. crassa and yeast polypeptides are in the same order (NH2-A-B-COOH). This arrangement is the reverse of the gene order characteristic of all prokaryotes that have been examined. N. crassa tryptophan synthetase has strong homology to the yeast TRP5 polypeptide (A domains have 54% identity; B domains have 75% identity), and somewhat weaker homology to the E. coli trpA and trpB polypeptides (A domains have 31% identity; B domains have 50% identity). The two domains of the N. crassa polypeptide are linked by a connector of 54-amino acid residues that has less than 25% identity to the 45-residue connector of the yeast polypeptide, although secondary structure analysis predicts both connectors would be alpha-helical. In contrast to the yeast TRP5 gene, which has no introns, the trp-3 coding region is interrupted by two introns 77 and 71 nucleotides in length. Both introns are located near the 5'-end of the gene and therefore not near the segment encoding the connector.

A total of 731 sera were collected from general hospital patients, divided into five distinct subgroups, and tested for the presence of immunoglobulin (IgG) to cytomegalovirus (CMV) and herpes simplex virus (HSV). The results indicate a high level of association between the two viruses, although HSV was found to be more prevalent in the earlier years of life, whereas CMV was acquired constantly throughout life. An increase in age was also accompanied by significantly higher antibody levels to both viruses. Individuals with antibody to HSV were significantly more likely to have antibodies to CMV, suggesting that these viruses are transmitted by similar routes (?saliva). In addition, 430 sera from 94 homosexual and 336 heterosexual males attending a clinic for sexually transmitted diseases were tested for antibody to CMV, HSV, and human immunodeficiency virus (HIV). Homosexual males were more likely to have antibody to CMV and HIV than were heterosexuals, but no difference was seen for HSV antibodies. The levels of CMV-specific IgG were significantly raised in homosexuals, compared with heterosexuals, but again no difference was seen for HSV antibodies as in the general hospital patients, however, individuals with HSV antibodies were significantly more likeoy to possess antibodies of CMV. However, the additional association of CMV antibodies with a homosexual lifestyle suggests that an alternative route for acquisitionof this virus exists (?semen). As raised levels of CMV antibodies, but not HSV antibodies, were found among homosexuals, this suggests that frequent CMV reinfections, rather than merely reactivation of latent herpes viruses, may be occurring.

N6-(delta2-isopentenyl)adenosine was found both as a component of tRNA and as the cytoplasmic mononucleotide in human leukemic lymphoblasts and myeloblasts from peripheral blood and bone marrow samples. This hypermodified nucleotide was also found in the tRNA and as a mononucleotide in human (MRC-5 and KB) and mouse (A9, FLV, LM, and RAG) cell lines. The relative amounts of this hypermodified nucleotide in the tRNA of the cell lines and the human leukemias were similar (the mean value being 0.06 +/- 0.03 mole % of the total tRNA nucleotide content); whereas the amounts occurring as the free cytoplasmic mononucleotide were more varied but still comparable (the mean value being 0.53 +/- .09 mole % of all cytoplasmic nucleotides) for all cells investigated with the notable exception of all normal, diploid cell lines under study (0.04 mole%). A possible relationship of the free cytoplasmic mononucleotide with the nucleotide in the tRNA for control of mammalian cell protein synthesis in vivo was investigated by addition of N6-(delta2-isopentenyl)adenosine to the culture medium. The exogenously added nucleoside caused inhibition of cell growth within 3 h and cell death within 36 h at concentrations as low as 0.4 muM. No comparable effects were seen when adenosine, adenine, or N6-(delta2-isopentenyl)-adenine were added to the cultures. The simultaneous presence of adenosine in cultures containing N6-(delta2-isopentenyl)adenosine did not alter the detrimental effects of the hypermodified nucleoside on cell growth even when the concentration of adenosine was 50-fold that of N6-(delta2-isopentenyl)adenosine. Addition of N6-(delta2-isopentenyl)adenosine to cell cultures caused within the first 6 h a significant reduction in the rates of RNA and protein synthesis; whereas DNA synthesis continued at a rate comparable to control and adenosine-treated cells for 18 h before decreasing.