[Penicillin-resistant pneumococci and the empirical use of penicillins in the treatment of community-acquired acute pneumonia]. The prevalence of penicillin-resistant pneumococci in our environment has raised questions about the effectiveness of penicillin as empiric treatment for community-acquired pneumonia cases. We followed prospectively all patients with community-acquired pneumonia from February 1989 through January 1990. We also reviewed retrospectively the treatment and evolution of all patients with confirmed pneumococcal pneumonia diagnosed between January 1988 and January 1990. A total of 115 patients with probable pneumococcal pneumonia were prospectively followed-up. Seventy-nine were treated with penicillin (benzyl- and aminopenicillin), and the remaining patients with macrolides, cephalosporin drugs or both. Five patients died (4%). There is no significant differences between mortality in penicillin-treated patients (2 cases) when compared to patients with other treatments (3 cases). Twenty-three patients have confirmed pneumococcal pneumonia. Among them, 8 (24%) had penicillin-resistant pneumococci (5 strains with MIC: 0.12-1 microgram/ml; 3 strains with MIC: 2 micrograms/ml). No differences were recorded regarding demographic data, predisposing conditions, underlying diseases, severity of pneumonia or the outcome of treatment between penicillin and non-penicillin treatment groups. Also, no differences were seen in clinical response and mortality when patients with pneumonia due to penicillin-resistant pneumococci treated with penicillin were compared to the ones treated with other drugs. In two patients, initially treated with erythromycin, progression of the pneumonia was recorded. Erythromycin resistant pneumococci (MIC greater than 8 micrograms/ml) were recovered from transthoracic needle biopsy. Both patients recovered well when beta-lactam antibiotics were prescribed.(ABSTRACT TRUNCATED AT 250 WORDS)


Retrovirus-mediated gene transfer as an approach to analyze neuroblastoma relapse after autologous bone marrow transplantation. Disseminated neuroblastoma is a malignancy of children often treated by intensive chemotherapy/radiotherapy followed by autologous bone marrow transplantation (ABMT). A high proportion of those treated subsequently relapse. It is unknown if relapse is a consequence of residual disease in the patient or of contaminating malignant cells remaining in the infused marrow, which, of necessity, is harvested and stored prior to ablative chemotherapy/radiotherapy. The assumption that residual cells in the infused marrow contribute to relapse has lead to the adoption of marrow purging prior to reinfusion. However, neither the necessity nor the efficacy of the procedure have been established. We now show how retroviral-mediated gene transfer using the LNL6 vector may resolve this issue. Clonogenic neuroblastoma cells in patient marrow can be transduced and the NEOR gene detected by observing individual neuroblastoma cell colony growth in G418, and by polymerase chain reaction (PCR) of individual colonies. Efficiency of transduction is between 0 and 13.5%. If marrow is exposed to LNL6 prior to infusion and marked cells are detected at the time of relapse, this would demonstrate that infused marrow contributed to disease recurrence. The technique could then be used to analyze the efficacy of marrow purging techniques. Since normal progenitor cells from these patients are also marked, the technique can be used to study factors that modify reconstitution and transducibility of infused marrow. Clinical studies using this approach have now begun.


Use of cell-free retroviral vector preparations for transduction of cells from the marrow of chronic phase and blast crisis chronic myelogenous leukemia patients and from normal individuals. Marrow cells were exposed to the LNL6 or G1N safety-modified variants of the N2 retrovirus, which contain the G418 bacterial resistance gene neo. The frequency of acquisition of the G418 resistance phenotype following exposure to LNL6 or G1N was compared among hematopoietic progenitor cells from the marrow of patients with chronic phase chronic myelogenous leukemia (CML), blast crisis CML, or from nonleukemic individuals. Under the conditions of our experiments, the myeloid committed progenitor cells from 3 of 6 nonleukemic individuals, 9 of 18 chronic-phase CML patients, and 2 of 4 blast crisis CML patients acquired resistance to at least 1 mg/ml G418 following incubation with cell-free supernatants from the PA317 LNL6 or PA317 G1N producer cell lines. Ten of the 32 colonies growing up in 0.8 mg/ml G418 from chronic-phase marrow exposed to LNL6 were shown to contain the neo gene by polymerase chain reaction (PCR) assay of DNA. These results were consistent with estimates of the transduction frequency based on acquisition of resistance to G418 as the number of colonies growing under G418 selection was always greater at 0.8 mg/ml G418 than at higher concentrations of G418 (1.0-1.4 mg/ml). The average transduction frequency at each G418 concentration (1.0, 1.2, and 1.4 mg/ml) in cells from blast crisis CML cells ranged from 2 to 14%, as measured by acquisition of G418 resistance. Chronic-phase CML showed slightly lower average frequencies of transduction (0.6-2.8% of the colonies are G418 resistant). The average transduction frequency of cells from nonleukemic marrow was as high as that seen from the marrow of chronic-phase CML individuals.(ABSTRACT TRUNCATED AT 250 WORDS)


High-efficiency gene transfer mediated by adenovirus coupled to DNA-polylysine complexes. Employment of recombinant viruses as gene transfer vectors is limited by constraints on the size and functional design of the genetic material to be transferred as well as potential safety hazards deriving from obligatory co-transfer of viral genetic elements. As an alternative strategy that capitalizes on the efficient cellular entry mechanisms of viruses, we have derived adenovirus-polylysine-DNA complexes whereby foreign DNA is transferred bound to the exterior of the virion. This linkage was accomplished utilizing an antibody bridge in which a monoclonal antibody was rendered competent to carry DNA by the attachment of a polylysine residue. Attachment of the antibody-polylysine to the virus was by virtue of the antibody's specificity for the virion. The resulting vector system mediates high-efficiency gene transfer to target cells in vitro. In addition, this vector design allows greatly enhanced flexibility in terms of the size and design of heterologous sequences that can be transferred. Since this strategy selectively exploits viral entry functions, which are independent of viral gene expression, the potential exists to derive vectors that avoid the hazards deriving from transfer of parent virus genome.


Expression of chimeric neo-Rev response element sequences interferes with Rev-dependent HIV-1 Gag expression. Recombinant plasmids containing reiterated human immunodeficiency virus type 1 (HIV-1) Rev response element (RRE) sequences were constructed to suppress Rev-dependent HIV-1 Gag expression. The mammalian expression vectors pMAMneo containing one, three, or six repeats of the RRE sequence were cotransfected with a HIV-1 HTLV-IIIB proviral DNA into HeLa cells. All three RRE expression plasmids reduced replication of HIV-1 with similar efficacy. Furthermore, the chimeric expression vector pCMV neoRRE6 x ----(containing six copies of the RRE sequence) was used to establish HeLa cell lines constitutively expressing RRE. A plasmid encoding a Rev-dependent HIV-1 p24 Gag protein was cotransfected with the wild-type Rev expression plasmid into three different RRE-expressing HeLa cell lines. p24 Gag protein production in the culture supernatants of the HeLaneoRRE cells was compared with two neo-expressing cell lines. Although all cell lines (HeLaneoRRE, HeLaneo) displayed similar transfection efficiencies, p24 Gag protein synthesis was markedly reduced in the RRE-expressing cell lines in comparison to the control cells.


Ex vivo gene therapy of familial hypercholesterolemia. Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by a deficiency in the receptor that clears low density lipoprotein (LDL) from the serum (reviewed in Ref. 1 and 2). Patients with one abnormal LDL receptor allele have moderate elevations in plasma LDL and suffer premature coronary artery disease (CAD). Approximately 5% of all patients under 45 who have had a myocardial infarction carry this trait. Patients with two abnormal LDL receptor genes (homozygous deficient patients) have severe hypercholesterolemia and life-threatening coronary artery disease in childhood. Strategies for treating patients with FH are directed at lowering the plasma level of LDL. In heterozygotes, this is accomplished through the administration of drugs that stimulate the expression of LDL receptor from the normal allele (2). This therapeutic approach is not effective in the treatment of homozygous deficient patients, especially those that retain less than 2% of residual LDL receptor activity. Partial amelioration of hyperlipidemia has been achieved in some homozygous deficient patients by diverting the portal circulation through a portacaval anastomosis (3) and by chronic plasmapheresis therapy (4). A more direct approach has been to correct the deficiency of hepatic LDL receptor by transplanting a liver that expresses normal levels of LDL receptor. Three patients that survived this procedure normalized their serum LDL-cholesterol (5-9). We have used an authentic animal model for FH, the Watanabe Heritable Hyperlipidemic rabbit (WHHL), to develop gene therapies for the homozygous form of FH (10-13). The WHHL rabbit has a mutation in its LDL receptor gene which renders the receptor completely dysfunctional (12) leading to severe hypercholesterolemia, diffuse atherosclerosis, and premature death. The potential efficacy of gene therapy for FH is supported by a series of studies we have performed in the WHHL rabbit in which we have achieved metabolic improvement (14-18). Liver tissue was removed from WHHL rabbits and used to isolate hepatocytes and establish primary cultures. A functional rabbit LDL receptor gene was transduced into a high proportion of hepatocytes using recombinant retroviruses, and the genetically corrected cells were transplanted into the animal from which they were derived. Transplantation of the genetically corrected, autologous hepatocytes was associated with a 30-40% decrease in serum cholesterol that persisted for the duration of the experiment (4 months, Ref. 18). Recombinant derived LDL receptor RNA was detected in liver for at least 6 months. There was no apparent immunological response to the recombinant derived LDL receptor.(ABSTRACT TRUNCATED AT 400 WORDS)


Spo0A activates and represses its own synthesis by binding at its dual promoters. The Spo0A protein of Bacillus subtilis controls the onset of sporulation by regulating transcription of various genes in both positive and negative manners depending on the promoters affected. The expression of the spo0A gene occurs from two promoters (Pv,Ps), separated by 148 bp, and transcription switches from Pv to Ps early in the sporulation program. DNase I footprint analysis of the spo0A promoter region revealed three distinct sites of Spo0A binding: -4 to +19 relative to Pv, -17 to +1 relative to Ps, and a region between Pv and Ps. The Pv region and the region between the two promoters was sufficient for repression of Pv. Induction of Ps also required these regions which are upstream of -52 relative to Ps. Mutant Spo0A proteins containing asp----asn mutations at asp10 and asp56 were inactive in repression of the abrB promoter in vivo yet still retained DNA-binding activity. The results presented are consistent with a model in which the phosphorylated form of Spo0A acts directly at its promoters to achieve induction of Ps and repression of Pv. These effects at the spo0A promoter were independent of the presence of the major kinase, KinA.


The interaction between Bacillus subtilis sigma-A (sigma A) factor and RNA polymerase with promoters. The P2 promoter from Bacillus subtilis sigma-A (sigma A) operon and the strong phi 29 phage G3b promoter were used to study their interactions with free sigma A and with RNA polymerase holoenzymes (E sigma A and E sigma 70). No binding of free sigma A to the tested promoters was observed, suggesting that the B subtilis free sigma A does not bind promoter by itself for the initiation of RNA transcription. Different footprints of B subtilis RNA polymerase holoenzyme (E sigma A) on the P2 and G3b promoters were detected. The footprint on the P2 promoter is mainly in the -10 downstream region of the bottom strand (noncoding strand) DNA and limited on the top strand (coding strand), whereas the footprints on both strands of the G3b promoter are very clear. These results suggest that the footprint regions of RNA polymerase on a promoter and the strength of its binding to the promoter depend on the properties of the specific promoter DNA sequence. It also suggests that the -10 and its downstream regions are more important than the -35 region for the formation of the E sigma A-P2 promoter open complex. Footprints of B subtilis E sigma A and E coli E sigma 70 on the same G3b promoter are very similar on the top strand but different on the bottom strand, with the footprint being about 17 bases wider (-4 to +13) in the case of E coli E sigma 70. Since this region contains most of the bases involved in promoter DNA melting, we suggest that E coli and B subtilis RNA polymerases have different efficiency in forming the open complex with heterologous promoter DNA during initiation of transcription.


The effect of supercoiling on the in vitro transcription of the spoIIA operon from Bacillus subtilis. The spoIIA operon codes for an alternative sigma factor which appears in the early stages of a sigma factor expression cascade during sporulation in Bacillus subtilis. We have used a single round in vitro transcription assay to probe requirements for transcription initiation at the spoIIA promoter. Core RNA polymerase or holoenzyme containing sigma A was reconstituted with sigma H protein and used to transcribe the spoIIA promoter. Formation of heparin resistant transcription initiation complexes required that the spoIIA template be supercoiled. Topoisomers of the spoIIA template were created and transcribed at various temperatures. Changes in the superhelicity of template DNA had a significant influence on the amount of transcription complexes formed at the spoIIA promoter.


Effect of mutant small, acid-soluble spore proteins containing cysteine or tryptophan on DNA properties in vivo and in vitro. Two derivatives of the alpha/beta-type small acid-soluble spore protein (SASP) SspCwt have been constructed, each containing a residue potentially useful for physico-chemical analysis of protein-protein or protein-DNA interactions. In one mutant protein (SspCtrp) residue 27 (Met) was replaced by Trp; in the second (SspCcys) residue 48 (Asn) was replaced by Cys. Both mutant proteins were expressed in Bacillus subtilis spores at levels similar to those of SspCwt, and SspCcys and SspCtrp restored ultraviolet light (UV) resistance and plasmid negative supercoiling in spores lacking major alpha/beta-type SASP to levels similar to those restored by SspCwt. While the purified mutant proteins bound more weakly to DNA than SspCwt, all three had the same relative affinity for different DNAs, ie poly(dG).poly(dC) greater than poly(dG-dC).poly(dG-dC) greater than pUC19, and purified SspCcys and SspCtrp gave the same pattern of DNase protected bands with pUC19 as SspCwt. Binding of SspCcys or SspCtrp to poly(dG).poly(dC) in vitro also prevented the formation of cyclobutane type cytosine dimers upon UV irradiation, as does binding of SspCwt. These data indicate that the two mutant proteins are extremely similar to SspCwt in their interaction with DNA, and thus may be useful in probing SASP-SASP and SASP-DNA interactions directly by physical or chemical techniques. Indeed, binding of SspCtrp to poly(dG).poly(dC) resulted in a 2.5-fold enhancement of the proteins Trp fluorescence.


Early spo gene expression in Bacillus subtilis: the role of interrelated signal transduction systems. The early spo genes are subject to a number of different control mechanisms. We found that at least one histidine kinase, SpoIIJ, is important for the expression of early spo genes but that two others, ComP and DegS, also affect sporulation, especially when SpoIIJ is absent. This indicates the existence of a signal transduction network which may gather information from several sources to feed into the sporulation pathway. Early spo gene expression is inhibited by overproduction of two response regulators, SpoOF and ComA. This effect is eliminated by the elevated presence of their cognate histidine kinases, SpoIIJ and ComP, respectively. This suggests that the unphosphorylated response regulators cause the inhibition of sporulation.


The spoIIN279(ts) mutation affects the FtsA protein of Bacillus subtilis. The spo-279(ts) mutation, originally thought to be located in the spoIIG operon of Bacillus subtilis, has been mapped in close proximity but outside of the spoIIG locus. This mutation defines a new gene, spoIIN, located midway between the spoIIG and the spoVE loci, and whose product is required for successful completion of the asymmetric septation step. The spoIIN locus was cloned using a combination of 'walking steps' upstream from the spoIIG region and hybridization screening of a bacteriophage lambda library. Sequencing of DNA fragments able to rescue the spoIIN279(ts) mutation revealed that the spoIIN locus is identical with the B subtilis counterpart of the Escherichia coli ftsA gene. After cloning the ftsA region from a strain containing the spoIIN279(ts) mutation we found that this mutation converts the ninth residue of the FtsA protein from serine to asparagine. The spoIIN279(ts) mutation, which is recessive, leads to filamentation during growth at 42 degrees C and causes defective formation of the sporulation septum at this non-permissive temperature. The FtsA protein is therefore required for proper cell septation, both during vegetative growth and sporulation. Possible additional roles of FtsA during sporulation are discussed.


Suppressors of a spo0A missense mutation and their effects on sporulation in Bacillus subtilis. The spo0A gene product of Bacillus subtilis is a transcriptional regulator that is required for the initiation of sporulation. It has not been possible to isolate mutations that suppress the sporulation defect caused by spo0A null mutations. We describe the isolation and characterization of mutations that suppress the severe sporulation defect caused by a spo0A missense mutation (spo0A9V). Two suppressor mutations, spa2 and spa4, have been characterized in combination with, and separated from, the spo0A9V mutation. Both were located in the carboxyl half of Spo0A, in the putative DNA binding, transcriptional activation region. spa2 was in codon 174, causing a leucine to arginine change (spo0A174LR), and spa4 was in codon 162 (of 267), causing a histidine to arginine change (spo0A162HR). spa2 and spa4 significantly restored sporulation to the spo0A9V mutant, however, the appearance of heat resistant spores was delayed relative to wild-type. When separated from spo0A9V, that is, as single mutations in spo0A, spa4 caused a delay in sporulation, while spa2 allowed apparently normal sporulation. The spa mutations caused interesting phenotypes when combined with other early sporulation mutations. spa2 suppressed the sporulation defect caused by spo0E11. This was most easily seen in spo0E11 abrB double mutants, which had a much more severe sporulation defect than the spo0E11 single mutant. That is, spo0E11 and abrB mutations caused a synthetic (synergistic) sporulation phenotype. Both the spa2 spo0A9V and the spa4 spo0A9V alleles greatly enhanced the sporulation defect caused by mutations in spoIIJ, spo0J and spo0K. The significance of these synthetic sporulation defects is discussed.


Intergenic suppression of stage II sporulation defects by a mutation in the major vegetative sigma factor gene (rpoD) of Bacillus subtilis. The Bacillus subtilis intergenic suppressor mutations crsA and rvtA, previously shown to restore sporulation competence to a variety of strains containing stage 0 sporulation defects, also suppress lesions in the stage II sporulation genes spoIIF, spoIIN and spoIIJ. They do not rescue sporulation in other stage II through stage V sporulation mutations. Cells containing spoIIN, spoIIF96 and spoIIJ::Tn917 mutations fail to transcribe spoIID, a late stage II gene. Introduction of crsA47 into spoIINts279, spoIIF96, or spoIIJ::Tn917 mutant backgrounds circumvents the need for the spoIIF, IIN, and IIJ products, restoring both expression of spoIID, and sporulation competence.


Protein filaments may initiate the assembly of the Bacillus subtilis spore coat. The Bacillus subtilis spore coat consists of three morphological layers: a diffuse undercoat, a striated inner coat and a densely staining outer coat. These layers are comprised of at least 15 polypeptides and the absence of one in particular, CotE, had extensive pleiotropic effects. Only a partial inner coat was present on the spores which were lysozyme-sensitive. The initial rate of germination of these spores was the same as for the wild type but the overall optical density decrease was greater apparently due to the loss of the incomplete spore coat from germinated spores. Suppressors of the lysozyme-sensitive phenotype had some outer coat proteins restored as well as some novel minor polypeptides. These spores still lacked an undercoat and germinated as did those produced by the cotE deletion strain. The CotE protein was synthesized starting at stage II-III of sporulation, long before the appearance of the coat on spores at stage IV-V. Despite its apparent hydrophilic properties, this protein was present in the crude insoluble fraction from sporulating cells. CotE was not solubilized by high or low ionic strength buffers not by detergents used for the solubilization of membrane proteins. Either 8 M urea or 6 M guanidine HC1 was required and dialysis against a low ionic strength buffer resulted in aggregation into long, sticky filaments. Both the CotE and CotT spore coat proteins appeared to be necessary for the formation of these filaments. Each of these proteins contains sequences related to a bovine intermediate filament protein so their interaction could result in an analogous structure.(ABSTRACT TRUNCATED AT 250 WORDS)


Use of a new integrational vector to investigate compartment-specific expression of the Bacillus subtilis spoIIM gene. The product of the spoIIM gene of Bacillus subtilis is required for complete septum migration and forespore engulfment during sporulation. To investigate whether expression of spoIIM is required in the forespore compartment of the sporangium, we have constructed a new integrational vector, pKSV7, which contains temperature-sensitive replication functions derived from pE194ts. The presence of the conditionally defective replication origin greatly stimulates plasmid excision when sporulation occurs at the permissive temperature. This facilitates the use of a genetic technique employed by Illing et al to distinguish genes whose expression must occur in the forespore from genes that may be expressed exclusively in the mother cell compartment. The results of the integration/excision experiments using pKSV7 support the conclusion that spoIIM must be expressed in the forespore. Biochemical analysis of forespore and mother cell fractions suggests that spoIIM is also expressed in the mother cell. The conditional integrational vector pKSV7 replicates at high copy number in E coli and allows the identification of inserts in the polylinker cluster by disruption of alpha-complementation and thus should be useful for other kinds of genetic manipulations in B subtilis.


Cloning and sequencing of the Bacillus megaterium spoIIA operon. The spoIIA operon of Bacillus megaterium has been cloned and the nucleotide sequence determined. The spoIIA sequence contains three open reading frames coding for putative proteins of 116 aa, 147 aa, and 253 aa; the first and the third genes are preceded by a ribosomal binding site. The genes are in the same order as those of B subtilis and B licheniformis. The deduced amino acid sequences of these three open reading frames show 78-92% homology with SpoIIAA, SpoIIAB and SpoIIAC of B subtilis and B licheniformis. Northern hybridization revealed that B megaterium also has two spoIIA transcripts, 1.77 kb and 2.92 kb, attaining maximum expression at t1 and t3, respectively. In addition, homology to a possible penicillin binding protein gene upstream and the first part of a spoVA operon downstream has been identified on the 3.34-kb fragment. The spoIIA and the downstream spoVA promoter regions are highly conserved among these three species. Sequence analysis of the spoVA promoter revealed a region upstream to the -35 that is highly conserved across Bacillus species.


A method for the determination of bacterial spore DNA content based on isotopic labelling, spore germination and diphenylamine assay; ploidy of spores of several Bacillus species. A reliable method for measuring the spore DNA content, based on radioactive DNA labelling, spore germination in absence of DNA replication and diphenylamine assay, was developed. The accuracy of the method, within 10-15%, is adequate for determining the number of chromosomes per spore, provided that the genome size is known. B subtilis spores were shown to be invariably monogenomic, while those of larger bacilli Bacillus megaterium, Bacillus cereus and Bacillus thuringiensis, often, if not invariably, contain two genomes. Attempts to modify the spore DNA content of B subtilis by altering the richness of the sporulation medium, the sporulation conditions (liquid or solid medium), or by mutation, were apparently unsuccessful. An increase of spore size with medium richness, not accompanied by an increase in DNA content, was observed. The implication of the apparently species-specific spore ploidy and the influence of the sporulation conditions on spore size and shape are discussed.


Evidence that recombination between reiterated sequences in the Bacillus subtilis chromosome does not occur via unequal crossing over. An experimental system was designed to permit the detection of recombination events occurring via unequal crossing over between sister bacterial chromosomes in Bacillus subtilis. It exploits the fact that during spore development, genetic and metabolic cooperation occurs between two different cell types, only one of which survives. During the early stages of sporulation, the two chromosomes of the developing sporangiole lie in the same cell and recombination between them is possible, in principle. Internal duplications flanking a selectable antibiotic-resistance gene have been introduced into the spoIIIC, spoIVA and spoVJ genes, whose correct expression in the mother cell (non-surviving compartment) is necessary for completion of spore development. After incubation in a sporulation-inducing medium in the absence of selective pressure, these strains sporulate at a low frequency and up to 30% of the progeny are Spo-. They result from mosaic sporangioles, in which only the chromosome segregated into the mother cell compartment of the developing sporangiole contains a reconstituted spo gene. In mosaic sporangioles generated by unequal crossing over between sister bacterial chromosomes, the insertionally inactivated spo gene, segregated into the pre-spore compartment, would carry an extra copy of the duplication initially present. Analysis of the products of 124 independent recombination events giving rise to mosaic sporangioles provided no evidence for the occurrence of unequal crossing over.


A Bacillus subtilis morphogene cluster that includes spoVE is homologous to the mra region of Escherichia coli. It is known that there is a strong similarity in amino acid sequence between the products of the Escherichia coli morphogenes ftsW (mra region at 2 min) and rodA (mrd region at 14 min) and the Bacillus subtilis SpoVE protein which is required for spore cortex formation. We show here that the predicted amino acid sequences coded for by the genes flanking spoVE are homologous to the products of the E coli genes murD and murG, which flank ftsW, and are involved in peptidoglycan biosynthesis. During vegetative growth and early stationary phase spoVE is cotranscribed with murD and murG in the form of very long polycistronic messages originating upstream of murD. However, this transcriptional activity is shut-off soon (approximately 1 h) after the cells enter stationary phase, and spoVE is then transcribed at two times during sporulation from its own promoter(s). Insertional in vitro mutagenesis of the region revealed that although murD and murG are essential for normal vegetative growth, spoVE is only required for sporulation: spoVE null mutants display a sporulation stage V phenotype indistinguishable by light microscopy from the phenotype conferred by the spoVE85 and spoVE153 alleles that originally defined the locus.


Molecular characterization of regulatory elements controlling expression of the Bacillus subtilis recA+ gene. Expression of the Bacillus subtilis recA gene is induced following DNA damage as well as during the development of the competent state. DNA damage-induction of the recA gene occurs by a RecA-dependent mechanism, whereas competence-induction occurs by a RecA-independent mechanism. To examine the molecular mechanisms that control the expression of the recA gene, a deletion analysis of the recA promoter region was performed. A regulatory region that is required for repression of recA expression was identified upstream of the recA promoter. Deletion of this regulatory region derepressed expression and abolished damage-induction of the recA promoter. Within this region are sequences similar to the consensus sequence that has been identified within DNA damage-inducible promoter regions of other B subtilis genes. Another regulatory region was identified that is required for the RecA-independent, competence-specific induction of the recA gene. Deletion of these sequences significantly reduced competence-induction of the recA promoter.


Haematospermia.

Haematospermia. Haematospermia, blood in the ejaculate, is a symptom which provokes great anxiety in patients due to fears of malignant or sexually transmitted disease. However, there is no evidence from the published literature to associate it with any serious pathology. The large series of cases indicate that investigation is unproductive and that patients do not develop serious disease even after prolonged follow-up. Patients presenting with haematospermia warrant a full physical examination, including rectal examination, but in the absence of physical signs they should then be strongly reassured. Further investigation is unnecessary. Coexistent urological symptoms should be investigated appropriately.


Herpes simplex virus infection in pregnancy and its management. Neonatal herpes simplex virus (HSV) infection is considered to be rare in the UK, affecting less than 3 per 100,000 live births, but the true incidence is probably higher due to under-reporting. In contrast, neonatal HSV infection is more common in the USA affecting 1 per 7500 live births overall. Infection in neonates is frequently serious and may be fatal.


Achievement of complete Bacillus subtilis microcycle sporulation by the addition of S-adenosylmethionine and phospholipids. In an attempt to find factors that may be responsible for the initiation of sporulation, a system in which the germination and outgrowth phases were separate was applied to Bacillus subtilis. Outgrowth of the germinated spores to only the primary singlet cells was followed in chemically defined medium. Addition of specific metabolites induced the primary singlet cells to sporulate via microcycle sporulation. Experiments are described that led to complete sporulation by the addition of diaminopimelic acid, S-adenosyl-L-methionine and phosphatidylethanolamine.


Pathophysiological concept of Haemophilus ducreyi infection (chancroid). Our knowledge concerning the pathogenesis of infection due to Haemophilus ducreyi is incomplete. In order to produce disease, H. ducreyi must presumably penetrate the skin of the external genitalia, colonize subcutaneous tissues, then produce tissue damage which results in ulcer formation. Penetration of the normal skin most likely occurs via minor abrasions. Adherence of H. ducreyi to different cell lines in vitro has been described, and might be mediated by adhesions such as pili or haemagglutinins. In addition, binding to extracellular matrix proteins has also been reported. Extracellular tissue-degrading enzymes were absent from broth culture supernatants of H. ducreyi. Such supernatants also failed to produce cytopathic effects with established or primary cell lines. Both live and heat-killed H. ducreyi organisms were able to produce lesions in a rabbit or a mouse model, although ulcer formation was dependent on viable H. ducreyi organisms in a recently introduced temperature-dependent rabbit model. With an excessive supply of iron, a more prolonged localized inflammatory disease effect was observed. Results derived from a subcutaneous chamber model demonstrated considerable changes in the expression of outer membrane proteins combined with antibody modulation during in vivo growth of H. ducreyi. These might be important factors for maintenance of infection in the human host particularly as these changes also occur in humans. Despite an increased knowledge of the pathogenesis of chancroid, important questions such as growth requirements, bubo-formation, role of cell-mediated immunity and ulcer formation are still unanswered. The application of molecular biological techniques in order to study these problems will be helpful.


Current smoking habits and genital infections in women. A study was undertaken to assess the relationship between current cigarette smoking and genital infections. Four hundred women attending a sexually transmitted disease clinic were the subjects of the study; of these 212 (53%) were cigarette smokers. In women under 20 years of age 70% were smokers. Women who smoked were more likely to have multiple partners and be in a lower socio-economic class or unemployed. The presence of genital warts was commoner in smokers. No association was shown between smoking and cervical inflammation or dysplasia. The findings suggest that cigarette smoking is a behavioural factor which should routinely be identified in the demographic details of women attending sexually transmitted disease clinics.


IgM-antibodies against T. pallidum detected in sera from mothers of stillborn babies in Mozambique by the solid-phase haemadsorption assay (SPHA). Serum samples taken at the delivery from 27 syphilitic mothers in Mozambique, 16 with stillborn babies and 11 with healthy babies were tested upon presence of IgM antibodies against T. pallidum by the solid-phase haemadsorption assay (SPHA). Fourteen out of the 16 serum samples from mothers with stillborn babies but only one out of the 11 samples from mothers with healthy babies were found positive by the SPHA test. Clinical signs indicative of syphilis are difficult to find in the Maputo area as there were more than 40,000 annual births and only five obstetricians when the study was carried out. It is believed that the findings may indicate those mothers who were serologically positive with the SPHA test had clinically active syphilis and that syphilis might be the cause or a contributory cause of death in 14 of the stillborn babies. The SPHA test was easy to perform and we recommend its adoption by laboratories with facilities to perform the TPHA test.


Chlamydia trachomatis antigen detection by Chlamydiazyme combined with Chlamydia Blocking Reagent verification. Several options exist for the detection of chlamydial infection in a routine laboratory setting. Enzyme immuno assay (EIA) technology offers rapid turn around of results and is less technically demanding than chlamydial cell culture. In addition, recently introduced EIA confirmatory reagents have the potential to improve the accuracy of EIA detection. We have evaluated one such confirmatory reagent (Chlamydia Blocking Reagent, Abbott Laboratories) to determine the accuracy of the Chlamydiazyme EIA with special regard to interpretation of low absorbance values. An initial series of 192 male urethral specimens showed that use of a lowered cut off level (absorbance value 0.05) compared with that recommended by the manufacturer increased sensitivity of the EIA from 0.73 to 0.83, thus motivating studies on this interpretative modification. Of 1101 EIA reactive specimens, 65% were determined to be chlamydia positive by the Chlamydia Blocking Reagent. The proportion of female cervical specimens that did not confirm positive was elevated compared with male urethral specimens, 43% vs. 5.7% respectively. In samples yielding absorbance from the recommended cut off level to 0.05 (approximately 50% below), the corresponding figures were 78% and 14% respectively. In 85 selected EIA reactive samples, examination by a direct immunofluorescence staining assay (DFA) (MicroTrak, Syva Inc.) revealed elementary bodies in 85% of 67 blocking test positive and in 24% of 18 blocking test negative samples. The possibility that Gram-negative bacteria were responsible for unconfirmed EIA reactive specimens was investigated using bacterial suspensions. While EIA reactivity was noted with several strains for Gram-negative bacteria, both the blocking reagent and DFA correctly verified the absence of chlamydial antigen.(ABSTRACT TRUNCATED AT 250 WORDS)


Factors associated with sexually transmitted diseases among prostitutes in Singapore. From June to December 1990, 806 prostitutes registered with the STD programme in Singapore for regular screening for sexually transmitted diseases (STD) were investigated for factors associated with STD incidence in the preceding year. The majority were foreigners (92.7% Malaysians and 3.1% Thais). Anal sex (0.4%) and intravenous drug use (0.9%) were rare. The overall STD incidence rate was 47.7 per 100. None was human immunodeficiency virus (HIV) positive. The crude and age-adjusted risk of STD was found to increase significantly with client load. An inverse relationship between condom use and STD risk was also observed. Mean condom use among clients was reported as 56.1% for spontaneous use and estimated as 75.4% following negotiation for condom use by prostitutes. Although the prostitutes negotiated for condom use with majority of the clients (85.5%) who did not use condoms spontaneously, they were successful with only about half of them (54.4%). Health education should be targeted at clients on the protective effects of condom use and at the prostitutes on skills in negotiating condom use.


Clinical study with recombinant interferon gamma versus interferon alpha-2c in patients with condylomata acuminata. A multi-centre, randomized, open-label trial was conducted to evaluate the safety and efficacy of recombinant interferon (rIFN) alpha-2c versus rIFN gamma in patients with recurrent or persistent condylomata acuminata (CA). Thirty-three such patients were treated either with 6 micrograms rIFN alpha-2c or with 0.1 mg rIFN gamma (both equivalent to 2 x 10E6 IU), single dose, subcutaneously 3 times a week for 6 weeks. In case of no complete clearance at week 10, a second course of treatment with the other type of rIFN was given. There was no significant difference in the complete clearance proportions at week 10 between the two treatment groups (3/16 vs 6/17). No relapses occurred in these patients during the 16 weeks' follow-up. Further clearances during the follow-up resulted in a total complete clearance proportion of 14/33 at the end of study. The treatment was well tolerated. Repeated interferon therapy has its place in treating persistent or recurrent condylomas.


[Clinical heterogeneity of the autistic syndrome: a study of 60 families]. Sixty families ascertained through a single proband, has helped to better define infantile autism as a heterogeneous group of disorders. Forty four patients showed a characteristic facio- auricular dysplasia. Twenty four of these, showed increased pyruvate and lactate and laboratory findings of metabolic acidosis i.e., anion gap above 18 mEq/L or serum bicarbonate below 21 mEq/L but only nine of these probands demonstrated reduction of plasma bicarbonate below 18 mEq/lt. Plasma amino acids in 17 probands and matched controls showed increased taurine with the rest of amino acids significantly (p less than 0.05) below the control level. Glutamate and aspartate were also significantly elevated (p less than 0.05; Student t-test). Segregation analysis in thirty four of these families which linked through at least one ancestral family name, suggested autosomal recessive inheritance (p = 0.20). Three out of eight probands who received megadoses of pyridoxine (Vitamin B6), subjectively gained in language abilities, affectivity and response to behavior modification therapy. Five autistic patients proved to have clinically defined syndromes: two with the Martin-Bell syndrome, and three girls affected respectively with the Rett syndrome, phenylketonuria and dicarboxylic aciduria.


Value of bacteriological screening of urine samples from HIV infected patients. A retrospective analysis of the results of bacteriological examination of all urine samples from adult HIV infected patients admitted to the City Hospital, Edinburgh during the year 1 April 1988 to 31 March 1989 was made to assess the usefulness of this investigation in HIV positive patients without clinical evidence of urinary tract infection.


[Serotyping of 48 isolates of Candida albicans: predominance of serotype A over B in Venezuela]. The study of serotyping of isolates of Candida albicans of clinical material obtained from different geographic areas of Venezuela suggest that serotype A is predominant over type B. These results are in relation with results obtained in other countries. Type A serotype is predominant in 57 to 100% in the areas studied. Only in 2 cities serotype B was isolated. Both serotypes of Candida albicans were observed in intertriginous, mucosal, inguinal and lung forms. These studies have allowed us to have information about the predominance of serotypes in certain areas of the country.


Spinal cord toxoplasmosis in a patient with human immunodeficiency virus infection. Spinal cord toxoplasmosis occurs rarely in the acquired immunodeficiency syndrome (AIDS). It usually presents as a space occupying lesion which makes differentiation from a neoplasm difficult. As a result, only 2 of the 5 previously reported cases have been diagnosed antemortem. We have incorporated the clinical details from one further case to describe the clinical features of this condition. The clarification of this clinical entity may allow earlier diagnosis of this rare condition in the future.


[Treatment of patients with panic disorder and an elevated urinary excretion of MHPG using imipramine or clonazepam]. The response to treatment with imipramine or clonazepam was evaluated in patients with panic disorder. These patients had a high urinary 3-methoxy-4-hydroxy-phenylglycol 24 h-excretion and normal plasma lactate levels. Both drugs were efficient as anti-panic agents, but clonazepam resulted more efficacious than imipramine by the scores of Hamilton Anxiety or Depression Scales. Moreover, clonazepam also reduced the anticipatory anxiety.


[Cytopathogenic effect of diphtheria exotoxin in cultured chick embryo fibroblasts]. One hundred fifty strains of Corynebacterium diphtheriae were studied from patients with symptoms of diphtheria and from healthy persons. 136 strains (90.67%) belonged to the mitis biotype, 12 (8.0%) and 2 (1.33%) to gravis and intermedius biotypes respectively. Toxigenicity was determined by traditional in vivo methods with rabbits and plaque immunodiffusion in KL-virulence medium. From the 136 mitis biotype strains studied, 130 (95.58%) showed positive toxigenicity by both methods and 11 strains (91.66%) from gravis biotype and 2 (100%) of intermedius biotype gave same results. The cellular cytotoxicity of the diphtheriae toxin was tested on culture of chicken embryo fibroblast and were positive in 132 (97.05%) strains of mitis biotype, and in all strains of gravis and intermedius biotypes. These results suggest that the cytotoxicity test seems to be a more sensitive method for detecting diphtheriae toxin production.


The solubility of volatile anaesthetics in water at 25.0 degrees C using 19F NMR spectroscopy. Anaesthetic concentration is very important for the quantitative treatment of anaesthesia theory. Traditionally concentration values have been derived from the water/gas partition coefficient. However, the values from many investigators show discrepancies. This study reports the accurate solubility of methoxyflurane (9.1 mM), halothane (18.0 mM), enflurane (11.9 mM) and isoflurane (13.5 mM) in water at 25.0 degrees C using 19F NMR spectroscopy. The method has advantages in that the dissolved molecule in solution can be separately quantified from undissolved anaesthetic. Saturated solutions of the anaesthetic agents were prepared in situ in a NMR tube to avoid pressure and temperature changes in the solution.


New fluorescent derivatives of cyclosporin for use in immunoassays. The synthesis of new fluorescent derivatives of cyclosporin is described and their affinity with the specific Sandoz monoclonal antibody investigated. Synthesis was carried out using cyclosporin-C-hemisuccinate as the starting material with monodansylcadaverine, 4-bromomethyl-7-methoxycoumarin, and 4-bromomethyl-6,7-dimethoxycoumarin as labels. After extraction the derivatives were purified by HPLC and their binding affinity with the monoclonal antibody evaluated by the Incstar Cyclo-Trac SP radioimmunoassay. All three derivatives showed good binding and it is suggested that they may be of use in an immunoassay for measuring cyclosporin.


Thermal behaviour and binary phase diagram of (S)-(+)-4,4'-(1-methyl-1,2-ethandiyl)-bis-(2,6-piperazinedione) (dexrazoxane), a cardioprotective agent, and of its (R)-(-)-enantiomer. The binary phase diagram of (S)-(+)-4,4'-(1-methyl-1,2-ethandiyl)-bis-(2,6-piperazinedione), 1 (dexrazoxane), a cardioprotective agent, and of its (R)-(-)-enantiomer, 2, has been investigated by differential scanning calorimetry (DSC); the equimolecular mixture of 1 and 2 corresponds to a racemic compound (racemate) whose melting point is higher than that of the enantiomers. Thermal behaviour (DSC) is examined and discussed in comparison with the data obtained by other physical methods (IR spectroscopy and X-ray powder diffraction).


Spectrophotometric determination of promethazine by flow injection analysis and oxidation by CeIV. A flow injection analysis (FIA) procedure is proposed for the determination of promethazine. The sample solution is directly injected into the carrier-reagent stream which comprises a solution of ceric ions in a sulphuric acid medium. The absorbance at 514 nm from the red colour developed by the oxidation of promethazine is measured. Effects of foreign substances have been investigated and the procedure has been applied to the determination of promethazine in a pharmaceutical formulation (tablets).


Determination of ampicillin in the presence of cloxacillin. A spectrophotometric method is described for the assay of ampicillin in the presence of cloxacillin in pharmaceutical preparations. It involves the reaction of hydrolysed ampicillin with formaldehyde in an acidic phosphate buffer (pH 2.5) and measurement of the absorbance of the product at 373 nm after dilution with 2 M sulphuric acid. The method is selective for ampicillin in the presence of cloxacillin. The absorbance had a linear relationship with concentration for the range studied (5-35 micrograms ml-1). By this method the ampicillin content of combined preparations of ampicillin and cloxacillin has been successfully determined in capsules, injections and a syrup.


Reversed-phase liquid chromatography of the opioid peptides--2. Quantitative structure-retention relationships and isocratic retention prediction. The ability of Snyder's theory of linear gradient elution to predict the starting isocratic reversed-phase LC conditions (k' = 4-10) for the opioid peptides was investigated. The errors in predicting the concentration of acetonitrile (phi) required to elute the peptides with a k' value of 4 were high, ranging from 13.5 to 38.1%. At k' = 10 the errors were generally reduced to less than 20%. This analysis was repeated with the same peptides after conversion to their fluorescent 1-cyanobenz[f]isoindoles (CBIs) by reaction with naphthalene-2,3-dicarboxaldehyde/cyanide. For the CBI derivatives, the errors in predicting the required concentration of acetonitrile for isocratic elution were markedly reduced and ranged from 0 to 14.3 for k' = 4 and 0 to 11.9% for k' = 10. The errors in the model in predicting the required isocratic mobile phase accurately were attributed to a mixed mechanism of retention involving solvophobic and silanophilic interactions and leading to non-linear relationships between log k' and phi. Even when the errors in predicting the required value of phi were relatively high, the Snyder approach was found to be very useful in predicting the initial starting conditions for the reversed-phase LC of the native opioid peptides as well as their fluorescence CBI derivatives.


Separation of reduced and oxidized glutathione in a pharmaceutical preparation by ion-interaction reversed-phase HPLC. A new method is presented for the simultaneous determination of reduced and oxidized glutathione by ion-interaction reversed-phase HPLC with octylamine orthophosphate as the interaction reagent, 5-microns Spherisorb ODS-2 as the stationary phase and UV detection at 230 nm. Lower limits of detection of 2.50 micrograms ml-1 have been achieved for both the oxidized and the reduced forms. The method has been applied in the analysis of a commercial pharmaceutical preparation.


High-performance liquid chromatographic determination of nifedipine and a trace photodegradation product in hospital prescriptions. A HPLC method is developed for the assay of nifedipine and trace amounts of a photodegradation product in pulverized tablets used in the preparation of hospital prescriptions. After liquid-liquid extraction, nifedipine and its photodegradation product are chromatographed using a reversed-phase system which involves UV detection at 254 nm and the use of two internal standards. The trace photodegradation product in pulverized tablets is determined along with nifedipine by the same procedure. The method is capable of determining 0.5-5.0 micrograms of nifedipine using p-nitronifedipine as the internal standard and 0.01-0.5 micrograms of the photodegradation product using 5-hydroxynifedipine as the internal standard. The standard error is found to not exceed 8.4%. The methods are useful for the study of the disposition or photostability of nifedipine in tablets and pulverized tablets.


LC assay for salmon calcitonin in aerosol formulations using fluorescence derivatization and size exclusion chromatography. A highly sensitive LC procedure was developed that utilizes fluorescence derivatization and detection coupled with size exclusion chromatography for the analysis of salmon calcitonin in salmon calcitonin aerosols. The LC procedure uses fluorescamine derivatization to label the primary amino groups of the peptide. The derivatization procedure is completely automated by an autosampler capable of pre-column mixing. Size exclusion chromatography is performed using a Supelco G2000 SWXL column. The method can be used to assay the amount of salmon calcitonin delivered per actuation of an aerosol unit. The procedure is simple, accurate, and precise and can detect as little as 2 ng ml-1 concentrations of salmon calcitonin.


Identification of caprolactam as a potential contaminant in parenteral solutions stored in overwrapped PVC bags. Semipreparative liquid chromatographic separation and subsequent off-line mass spectrometry have revealed caprolactam as a new contaminant in intravenous solutions. The content of the lactam was found to be 1.2-15.0 mg l-1 determined by liquid chromatography. Contamination is attributed to migration of caprolactam from the protecting plastic envelope through the PVC barrier and into the intravenous solution. Migration occurs during the final heat sterilization process.


On-line determination and resolution of the enantiomers of ketoprofen in plasma using coupled achiral-chiral high-performance liquid chromatography. High-performance liquid chromatography (HPLC) using a column-switching technique has been applied to the on-line determination and resolution of the enantiomers of ketoprofen (KPF) as an acidic model compound. The system incorporates a mobile phase conversion stage (LC-2), which has a dilution tube and a trapping column, between the achiral chromatography stage (LC-1) and the chiral chromatography stage (LC-3). KPF in plasma was separated from plasma components and was determined with the aid of an internal standard in LC-1. The eluate containing KPF was selectively transferred to LC-2, where the eluate was adequately diluted with a new mobile phase by using the dilution tube to reduce the influence of the mobile phase from LC-1, and KPF was reconcentrated on the trapping column. Then the KPF enantiomers were resolved in LC-3 after column-switching. This system is accurate and rapid compared with conventional HPLC. Very high sensitivity could be achieved with our system when a microbore ovomucoid column was employed in LC-3. Incorporation of LC-2 allows the most favourable mobile phases for LC-1 and LC-3 to be used independently. This method is greatly superior to usual column-switching HPLC.


Photolysis of cyanocobalamin in aqueous solution. Cyanocobalamin is photolysed in aqueous solution to produce hydroxocobalamin. The kinetics of photolysis has been studied at pH 1-12 using a newly developed spectrophotometric method for the simultaneous determination of the two compounds at 550 and 525 nm or 361 and 351 nm. Cyanocobalamin follows zero-order kinetics at the pH values studied and the rate is catalysed by both hydrogen and hydroxyl ions. The log k-pH profile indicates that cyanocobalamin has maximum stability near pH 7. The cationic species appears to be more susceptible to photolysis than the neutral form. The rate constant for the reaction at pH 1 is 1.32 x 10(-7) mol l-1 min-1 compared with 0.050 x 10(-7) mol l-1 min-1 at pH 7.


Radioimmunoassay for ubenimex in human serum. Anti-ubenimex antibody was produced by immunization of rabbits with ubenimex-bovine serum albumin (BSA) conjugate which was synthesized by coupling ubenimex directly to BSA using 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide. The resulting antibody exhibited little cross-reactivity with p-hydroxyubenimex or (2S,3R)AHPA, (2S,3R)-3-amino-2-hydroxy-4-phenylbutanoic acid present in human serum as metabolites. Ubenimex in human serum could be measured selectively without prior drug extraction and purification. The standard curve prepared using the present assay method gave good linearity in the range 0.25-25 ng per assay tube. The detection limit was 125 pg per assay tube. The concentrations of ubenimex in human serum determined by the present method were in good agreement with those obtained by GC-SIM. The present method with its high sensitivity and specificity may be a valuable tool for the study of the pharmacokinetics of ubenimex.


An automatic data reduction and transfer method to aid pattern recognition analysis and classification of NMR spectra. A method of automatically generating reduced NMR data and transferring it between computers is proposed. These data can then be used as descriptors for input to non-parametric statistical routines for classification of the samples.


Synthesis and analysis of O,O'-dicarboxylate (dibenzyl) bispilocarpates as possible prodrugs of pilocarpine. As a part of a series of studies to develop prodrug derivatives of pilocarpine, the O,O'-succinyl (dibenzyl), O,O-adipoyl (dibenzyl), O,O-fumaryl (dibenzyl), and O,O-terephthaloyl (dibenzyl) bispilocarpate fumarates were synthesized as a new class of pilocarpine prodrugs. The compounds were prepared from pilocarpic acid benzyl monoester by coupling two pilocarpic acid benzyl monoesters together with spacer chains by usual esterification methods. Liquid chromatography, thermospray liquid chromatography-mass spectrometry, high-resolution mass spectrometry, and NMR spectroscopy were applied to the identification and the purity evaluation of the synthetic products.


Competitive binding assay for quantitative determination of GM1 ganglioside in plasma and cerebrospinal fluid. A competitive binding assay for the quantitative determination of GM1 ganglioside is described. After extraction from biological fluids, GM1 was incubated with a known amount of cholera toxin B-subunit conjugated with horseradish peroxidase, and exposed to GM1 adsorbed onto polystyrene microwells. Since GM1 in solution blocks the binding of toxin B-subunit to GM1 adsorbed onto the solid phase, enzyme activity serves as a reciprocal measure of GM1 concentration in the sample. The assay was used to determine the basal level of GM1 in plasma and cerebrospinal fluid in different populations.


A selective and sensitive kinetic method for the determination of procaine and benzocaine in pharmaceuticals. A simple stopped-flow method for the determination of procaine and benzocaine which is suitable for their routine analysis in pharmaceutical samples is reported. The method is based on a condensation reaction between each compound and 4-dimethylaminocinnamaldehyde, which yields coloured compounds and which are monitored spectrophotometrically. The calibration graph generated is linear over the range 0.5-20 micrograms ml-1 (RSD 0.73%) for procaine and 0.5-15 micrograms ml-1 (RSD 0.40%) for benzocaine, and the selectivity is very high in both cases. The proposed methods were satisfactorily applied to the determination of the two drugs in various pharmaceutical samples. A procedure for resolution of procaine-benzocaine mixtures in mass ratios between 15:1 and 1:7 with a precision of ca 1% was developed in order to determine one compound in the presence of the other with no interference.


A safer methylation procedure with boron trifluoride-methanol reagent for gas chromatographic analysis of ritalinic acid in urine. Ritalinic acid (RITA), the major metabolite of methylphenidate (Ritaline) is extracted with a solid-phase C8 column. Following elution, methylation of the carboxylic group is performed with boron trifluoride-methanol as a reagent. The methyl ester of RITA which structurally corresponds to the parent compound methylphenidate is reextracted at pH 9-11. Each step of the analytical method has been systematically studied, particularly the parameters governing the esterification reaction with BF3-methanol. The recovery from the overall method is 96%. The limit of detection is 0.05 micrograms ml-1 with GC-NPD for a 10-ml urine sample. The method has been successfully applied to the detection and quantitation of RITA in urine specimens. The specificity of the methylated RITA peak has been verified by GC-MS in scan mode. Use of diazomethane or sulphuric acid is thus replaced by the use of a less hazardous reagent.


Collaborative study of the analysis of chlortetracycline hydrochloride by liquid chromatography on polystyrene-divinylbenzene packing materials. A previously established method for the analysis of chlortetracycline by liquid chromatography using polystyrene-divinylbenzene stationary phases was examined in a multicentre study involving four laboratories and a total of 12 columns. Three chlortetracycline hydrochloride samples were analysed. The main component and the impurities were determined. An analysis of variance, treating each column as a different laboratory, showed absence of consistent laboratory bias and presence of significant laboratory-sample interaction. Estimates for the repeatability and reproducibility of the method, expressed as relative standard deviations of the result of the determination of chlortetracycline hydrochloride, were calculated to be 0.7 and 1.2%, respectively. When the analysis of variance was performed using only the results obtained on the wide pore (1000 A) stationary phases, the laboratory-sample interaction strongly decreased. It is therefore proposed to use such materials for the analysis of chlortetracycline.


Fundamental studies in reversed-phase liquid-solid extraction of basic drugs; II: Hydrogen bonding effects. Anomalies in the elution order of the beta-blockers atenolol and propranolol on reversed-phase liquid-solid extraction cartridges have been studied. The hydrogen bond acceptor properties of the polar ring substituent in atenolol was found to result in greater retention than would be expected from the reversed-phase or cation-exchange mechanisms alone. This hydrogen bonding interaction could be attenuated by inclusion of a strong hydrogen bond acceptor in the eluent or more successfully by increasing the eluent water content. The conclusions from this work have been used to explain previously reported anomalies in the solid-phase extraction of polar basic drugs.


Chromatographic analysis of 1,3-bis(dimethylamino)isopropyl 4-chlorophenoxyacetate dihydrochloride, a new CNS stimulant. A reversed-phase liquid chromatographic method is developed for the assay of the new CNS stimulant, 1,3-bis(dimethylamino)isopropyl 4-chlorophenoxyacetate dihydrochloride (BCE-001), and its main contaminant, 4-chlorophenoxyacetic acid (PCPA). In the first place the possible reactions of BCE-001 with mobile phase components are investigated and it is found that BCE-001 is readily hydrolysed in water and undergoes transesterification in methanol or ethanol. The methyl ester is formed rapidly and quantitatively so that BCE-001 can be assayed as methyl 4-chlorophenoxyacetate. PCPA is formed as a result of the hydrolysis of BCE-001 during the sample pretreatment and chromatographic separation and this causes an overestimate of the PCPA impurity in the bulk drug. An effective method is developed to prevent the hydrolysis of BCE-001 during sample pretreatment.


Determination of sertaconazole nitrate, a new imidazole antifungal, by high-performance liquid chromatography. A high-performance liquid chromatographic method is developed for the determination of bulk sertaconazole nitrate and related compounds (potential impurities and degradation products) as well as a sertaconazole nitrate cream formulation. A 10-microns Spherisorb CN column is used along with a mobile phase consisting of acetonitrile and aqueous 0.01 M sodium phosphate (37:63, v/v). The sertaconazole nitrate peak is monitored at a wavelength of 260 nm; the retention time being 19.3 min. The detector response for sertaconazole nitrate is linear over the concentration range from 64 to 96 micrograms ml-1. The method is found to be sufficiently selective for the reliable determination of related compounds, FI-7001, FI-7009 and FI-7011, as indicated by same-day and between-day relative standard deviations (RSD) for replicate assays of 1.72% (n = 9) and 2.17% (n = 24), respectively. The application of this method to a cream formulation of sertaconazole nitrate is found to give a mean percentage recovery of 99.4% with RSD of 1.14% (n = 9); none of the cream vehicle peaks are found to interfere with the determination of sertaconazole nitrate.


Ion trap detector--capillary gas chromatography of valproic acid and its mono-unsaturated metabolites in serum using methyl ester derivatives. A quantitative method was developed for valproic acid and five of its mono-unsaturated metabolites using capillary gas chromatography-mass spectrometry with selected ion monitoring. The method was applied to serum and all metabolites were measured in a single run. Methyl esters were synthesized as the derivatives suitable for gas chromatography. Calibration curves were found to be linear and the sensitivities in the order of 0.1 micrograms ml-1. Patients' data are presented. By this method it is possible to separate the stereoisomers of 2-n-propyl-2-pentenoic acid and of 2-n-propyl-3-pentenoic acid.


Differentiation of linearly correlated noise from chaos in a biologic system using surrogate data. Experimental time-series of human H-reflexes were analyzed for the presence of fractal structure or deterministic chaos. Surrogate data sets consisting of stochastic time-series with preservation of selected properties of the experimental time-series were used as mathematical controls. Artifacts generated during the analysis of the experimental data are identified, and shown to be due to linear correlation in the original time-series. The method is simple and generally applicable to the non-linear analysis of time-series from any experimental system.


4-Bromomethyl-7-methoxycoumarin and analogues as derivatization agents for high-performance liquid chromatography determinations: a review. A major part of modern analytical problem solving deals with the trace level determination of organic compounds and contaminants in biomedical, food and environmental samples. In the analysis of these samples chromatographic techniques play a predominant role. Unfortunately, however, even the combined force of an efficient separation plus a sophisticated mode of detection does not always create sufficient selectivity and/or sensitivity for the final goal to be attained. In such cases, special attention has to be devoted to derivatization or conversion of the analyte(s) of interest (for improved detection selectivity and/or sensitivity) and sample pretreatment (for trace enrichment and clean-up). The above is especially true when, as is often the case today, relatively polar drugs, endogenous compounds, additives or environmental pollutants and/or their (bio)-degradation products have to be determined. For such classes of compounds high-performance column liquid chromatography (HPLC) generally is the preferred method of separation. Reversed-phase HPLC with fluorescence detection is a powerful means of analysis for compounds which possess native fluorescence. They are, however, relatively few in number. In order to make the method useful for a much wider range of analytes, one can therefore resort to derivatization (labelling) or other means of analyte conversion to obtain highly fluorescent reaction products, which can then be detected with the required selectivity and sensitivity. 4-Bromomethyl-7-methoxycoumarin is often used as fluorescent label for the determination of compounds possessing a carboxylic group. About 8% of the biologically interesting analytes--ranging from polar amino acids and peptides to apolar fatty acids--possess such a group.(ABSTRACT TRUNCATED AT 250 WORDS)


Evidence for a generalized Laguerre transform of temporal events by the visual system. It is generally assumed that the early visual processing is constituted by a set of filters operating in parallel. In this respect the visual system performs a transform, generating a code of the characteristics of the input signal. Recently, it has been suggested that the coding of the spatial characteristics by the visual system can be described by a Hermite transform (Martens, 1990a, b). It was also suggested that a three-dimensional Hermite transform can be used to code spatiotemporal events. In contrast to this latter suggestion, we argue that the coding of temporal events takes the form of a generalized Laguerre transform. We review psychophysical evidence supporting this hypothesis.


A study of chloroquine and desethylchloroquine plasma levels in patients infected with sensitive and resistant malaria parasites. This study was carried out on 63 patients in the town of Gadaref in eastern Sudan; each patient was given the standard therapeutic dose of chloroquine (CQ). Plasma levels of chloroquine and its major metabolite desethylchloroquine (DCQ) were measured by means of a high-performance liquid chromatographic method (HPLC) in patients infected with sensitive (sensitive group) and resistant (resistant groups) strains of Plasmodium falciparum. The ratios of chloroquine to desethylchloroquine (CQ/DCQ) in different groups were calculated and the results obtained were compared and correlated with the degree of parasitaemia. The statistical analysis of the results showed that the plasma content of CQ and the CQ/DCQ ratio in the majority of the patients fall within the normal mode of distribution. A small group of patients showed a deviation from the normal mode by having a rather high CQ plasma level and a high ratio of CQ/DCQ. The mean plasma levels of CQ and the CQ/DCQ ratio in the sensitive group was found to be higher than that in the resistant groups. However, these differences were found to be not significant. Correlation tests showed that the levels of CQ and the CQ/DCQ ratios increase with the increase of the degree of parasitaemia in the sensitive group but decrease with the increase of parasitaemia in resistant groups.


[Hypocaloric peripheral parenteral nutrition in postoperative patients (Proyecto Europan)]. Hypocaloric peripheral parenteral nutrition (HPPN) appears to be indicated in patients with moderate malnutrition subjected to a short period of fasting following surgery. Our objective is to determine whether or not the intake of hypocaloric parenteral solutions containing aminoacid is useful in postsurgical patients, by means of a study of different nutritional evaluation parameters. A study was performed on 35 postsurgical patients who fulfilled at least 2 of the following criteria indicating malnutrition: 1) albumin levels less than 3 g/dl; 2) prealbumin levels less than 21 mg/; 3) body weight less than 95% of ideal weight. The patients were divided into 3 groups: Group I consisted of 15 patients treated with conventional fluid therapy, Group II consisted of 10 patients treated with nutritional support based on glucose and aminoacid and Group III was comprised of 10 patients also treated with a nutritional therapy based on glycerol and aminoacid. The most significant data included a rapid recovery of short term proteins (prealbumin and retinol-binding protein) and a less negative nitrogen balance 5 days afterwards in both the glucose and glycerol groups, both of which were statistically significant. With regard to complications studied, there was a greater incidence of suture dehiscence in the control group than in the groups under treatment (13.3% compared to 50%). Our conclusion is that HPPN is a valid nutritional support measure in postsurgical patients in certain clinical situations and circumstances, although further controlled, randomized studies should be considered, during both the pre and post operative periods of these patients in order to clearly indicate how and when nutritional therapy should be applied.


[Nickel content of parenteral nutrition solutions]. Determination of the nickel content in 49 commercial solutions used in parenteral nutrition, by means of atomic absorption spectroscopy in a graphite kiln. Important differences were found between the different components (less than 5 to 23.000 ppb). The largest concentrations of Ni were found in sodium chloride, phosphate salts, 10% calcium chloride and 2M potassium chloride, whereas the aminoacid, glucose and lipid solutions contained the smallest concentrations. The amount of Nickel in the preparations varied depending on the supplier and different salts of the compound itself. The Nickel intake in 2 TPN solutions with high and low concentrations of Ni (827 and 270 micrograms respectively) was also analyzed, and this intake was higher than the estimated amount absorbed in the diet. In the case of the TPN with a high Nickel content, this was higher than the level detected as being the cause of sensitivity reactions in patients sensitive to Nickel.


Disambiguating ambiguous figures by a model of selective attention. Ambiguous figures are visual stimuli which are interpreted multiply by the human visual system. A model is proposed which disambiguates the ambiguous figures. The model was formulated based on the characteristics of visual information processing, accompanied with selective attention. In the ambiguous figure "my husband and my father-in-law", it was necessary to simulate visual information processing so that attention was directed to the multiple features in the figure to disambiguate the ambiguous figure. Pictures, obtained from the model, were examined as to whether they were interpreted unambiguously or not. Results show that the model, simulated selective attention, can disambiguate the ambiguous figures. This suggests that the image per se, viewed through selective attention, becomes unambiguous before the figure is interpreted in the higher level. Results also show that the computer simulation of selective attention would make it possible to examine factors affecting the initial interpretation of the figure.


Reduction of a Hodgkin-Huxley-type model for a mammalian neuron at body temperature. Hodgkin-Huxley-type models mimic the electrical behavior of excitable membranes quite realistically. However, inclusion of many different ionic channels into such a model yields a highly complex set of differential equations. In this paper a reduction of a "full" Hodgkin-Huxley-type model based on voltage-clamp data from small rat neurons in the supraoptic nucleus area is introduced. It was found that two of the ionic channel gating variables of the full model preserved a rather close relationship during simulations. This allowed to express one of these gating variables in terms of the other one thus reducing the number of differential equations the model is based on. The behavior of the reduced model was very similar to that of the full model. In particular, important physiological features as spike shape and constant-input-to-interspike-interval relationship were (almost) identical in the full and the reduced model.


Unsupervised clustering and centroid estimation using dynamic competitive learning. In this paper, an unsupervised learning algorithm is developed. Two versions of an artificial neural network, termed a differentiator, are described. It is shown that our algorithm is a dynamic variation of the competitive learning found in most unsupervised learning systems. These systems are frequently used for solving certain pattern recognition tasks such as pattern classification and k-means clustering. Using computer simulation, it is shown that dynamic competitive learning outperforms simple competitive learning methods in solving cluster detection and centroid estimation problems. The simulation results demonstrate that high quality clusters are detected by our method in a short training time. Either a distortion function or the minimum spanning tree method of clustering is used to verify the clustering results. By taking full advantage of all the information presented in the course of training in the differentiator, we demonstrate a powerful adaptive system capable of learning continuously changing patterns.


[Effect of total parenteral nutrition on bone metabolism]. Total parenteral nutrition (TPN) for a prolonged period of time can be associated with bone pain and osteomalacia. We performed a study on the phosphorus/calcium metabolism and serum levels of osteocalcin (BGP), a protein proposed as constituting the bone turnover index in 31 patients receiving TPN (age 57 +/- 14 years, 22 males and 9 females) diagnosed as suffering from pathology of the digestive tract or geno-urinary pathology. The duration of the TPN was from 9.1 +/- 6.6 days (range 2-31 days). We observed and increase of FA (178 +/- 101 U/l), with a significant decrease of BGP (2.2 +/- 2.0 ng/ml vs. 3.7 +/- 1.3 ng/ml in controls; p less than 0.001). Serum levels of phosphorus and calcium corrected according to proteins were within normal limits. Hypercalciuria was detected in the urine (328 +/- 278 mg/24 hours), and phosphaturia (607 +/- 522 mg/24 hours). Based on the BGP results, we can conclude that patients subjected to TPN for a short period of time undergo a decrease in bone turnover.


A coupled pacemaker-slave model for the insect photoperiodic clock: interpretation of ovarian diapause data in Drosophila melanogaster. A coupled circadian oscillator model for the insect photoperiodic clock is described which consists of a hierarchically arranged pacemaker and slave. The pacemaker is self-sustained, temperature compensated, and entrainable by the light cycle; the slave is a damping oscillation receiving entrainment from two sources, from the pacemaker via a coupling factor, and also directly from the light. The damping slave oscillation is seen as the "photoperiodic oscillator", equivalent to that proposed earlier by Lewis and Saunders (1987). The present simulations describe the effect of the strength of the coupling factor between hypothetical short- and long-period pacemaker oscillations (modelled on the "clock" mutants perS and perL2 in Drosophila melanogaster) and a slave oscillation with a period of about 24 hours. The output is presented in terms of photoperiodic response curves and Nanda-Hamner, or resonance, plots. With a high coupling strength, the pacemakers strongly entrain the slave, but with a low coupling strength the slave's properties are more evident. The model is presented as a possible explanation for recent ovarian diapause data in D. melanogaster "clock" mutants (Saunders 1990), but also as a more general model for the role of the insect circadian system in seasonal time measurement.


Two and three dimensional reductions of the Hodgkin-Huxley system: separation of time scales and bifurcation schemes. We study two different two-dimensional reductions of the Hodgkin-Huxley equations. We show that they display the same qualitative bifurcation scheme as the original equations but overestimate the current range where periodic emission occurs. This is essentially due to the assumption that the evolution of the sodium activation variable m is instantaneous with respect to the dynamics of the variables h and n, an hypothesis that breaks down at high values of the injected current. To prove this point we compare the current-amplitude relation, the current-frequency relation, and the shapes of individual spikes for the two reduced models to the results obtained for the original Hodgkin-Huxley model and for a three-dimensional model with instantaneous sodium activation. We show that a more satisfying agreement with the original Hodgkin-Huxley equations is obtained if we modify the evolution equation for the potential by incorporating the prominent features of the dynamics of m.


Information maintenance and statistical dependence reduction in simple neural networks. This study compares the ability of excitatory, feed-forward neural networks to construct good transformations on their inputs. The quality of such a transformation is judged by the minimization of two information measures: the information loss of the transformation and the statistical dependency of the output. The networks that are compared differ from each other in the parametric properties of their neurons and in their connectivity. The particular network parameters studied are output firing threshold, synaptic connectivity, and associative modification of connection weights. The network parameters that most directly affect firing levels are threshold and connectivity. Networks incorporating neurons with dynamic threshold adjustment produce better transformations. When firing threshold is optimized, sparser synaptic connectivity produces a better transformation than denser connectivity. Associative modification of synaptic weights confers only a slight advantage in the construction of optimal transformations. Additionally, our research shows that some environments are better suited than others for recording. Specifically, input environments high in statistical dependence, i.e. those environments most in need of recoding, are more likely to undergo successful transformations.


Gamma/delta receptor-expressing T-cell clones from a cutaneous T-cell lymphoma suppress hematopoiesis. Recently we described a cutaneous T-cell lymphoma expressing the gamma/delta T-cell receptor [5]. The patient suffering from this lymphoma showed low numbers of myeloid and T cells in peripheral blood, while B and NK cells were relatively increased. In vitro culture of the patient's bone marrow (BM) cells revealed a significant suppression of myeloid/monocyte colony formation (GM-CFU) compared with normal controls. This was not due to infiltration of the BM with lymphoma cells. We speculated that a soluble factor either secreted or induced by the lymphoma cells might be responsible for the marked suppression of hematopoiesis in this patient. From a skin biopsy with infiltrating gamma/delta T-lymphoma cells we established T-cell clones bearing the gamma/delta T-cell receptor and resembling the phenotype of the lymphoma cells. The supernatant (SN) of these gamma/delta T-cell clones reduced the number of colonies in a CFU-GM assay (using normal control BM) in comparison to SN of alpha/beta T-cell clones established from the same biopsy. This suppression was seen mainly on day 7 of culture and was not neutralized by the addition of placenta-CM. The main mediator of this suppression seems to be IFN-gamma, since it was detectable in high amounts in the SN of these gamma/delta T-cell tumor clones as well as in the serum of the patient. In addition, anti-IFN-gamma antibodies can reverse the T-cell SN-mediated suppression of CFU-GM. We conclude that high serum levels of interferon-gamma, which is secreted in high amounts from gamma/delta T-cells grown from a biopsy of a cutaneous lymphoma, can suppress hematopoiesis.


[Epidemiology of parenteral nutrition during 5 years of follow-up by a nutrition team]. The purpose of this retrospective study is to ascertain the physiopathological characteristics of patients on parenteral nutrition (PN), the types of diet used and duration of treatment. Presentation of epidemiological results and evolution of 637 adults receiving PN after a five-year period of nutritional follow-up, conducted by a Nutrition Team (NT) in hospital. For the purpose of this study, we used the Follow-Up charts of all the patients treated with PN from 1986-1990. The results obtained lead us to affirm that most patients suffered a malignant gastrointestinal process, that the duration of the PN was reduced significantly during the five-year term, mainly using dietary protocols, and that there was a gradual increase in preparations of nutrient units subjected to controls compared to the total prepared by the Pharmacy Department.


All-trans retinoic acid in acute promyelocytic leukemia: preliminary results in Cuba. Seven patients with acute promyelocytic leukemia (APL) were treated with all-trans retinoic acid (ATRA). Five (71.4%) achieved complete remission (CR). Most side effects were transient and well tolerated. Hyperleukocytosis was the major adverse effect. These observations confirm the efficacy of ATRA for inducing CR in APL.


[Nutritional support in laryngectomized patients]. Pathologies involving tumours of the neck are an important group of neoplasias. The tumours themselves are often the cause of malnutrition, and this may be aggravated in these patients by other factors such as: excess of alcohol and cigarettes, anatomical location of the tumour next to the digestive tract and the application of cyto-reducing treatment applied previously. The main purpose of the present study is to analyze the possible effect of hospital diets during the postoperative period in laryngectomized patients, on the development of malnutrition. For this purpose 49 patients were studied, all diagnosed consecutively during the last year as suffering from epidermoid carcinoma of the larynx, subjected to 24 total laryngectomies, 4 partial laryngectomies and 21 supraglottic laryngectomies. A conventional diet was administered at random to 21 patients, and a commercial enteral diet to the remaining 28. The most important conclusions were as follows: a) high incidence of harmful habits, b) positive nutritional state of the patients upon admittance, c) high incidence of malnutrition one week after the operation, which was maintained virtually in the same proportion to the time spent in hospital, d) the lack of influence of the nutritional state by type of diet administered, e) the possible influence on malnutrition by type of surgery performed, location of the tumour (pyriform sinus) and high incidence of complications usually developed.


Oxidative damage of erythrocytes infected with Plasmodium falciparum. An in vitro study. The extent of reduced glutathione, activity of glutathione peroxidase, amount of membrane lipid peroxidation products, and the extent of hemoglobin release from host erythrocytes during in vitro Plasmodium falciparum growth was studied. Highly synchronized parasite cultures were studied to examine the alterations caused by different growth stages of the parasite. There was a moderate increase in the reduced glutathione content as the parasite matured, which was significant only in schizont-rich erythrocyte lysates (p < 0.05) whereas the activity of glutathione peroxidase was significantly low in all the parasitized red blood cells (ring-infected RBC, p < 0.005; trophozoite- and schizont-infected RBC, p < 0.001). The lipid peroxidation product, malonyldialdehyde, of the host red cells increased gradually to more than fourfold in schizont-rich cells as compared with normal erythrocytes (p < 0.001). The hemoglobin release from cultured cells was significantly higher in all parasitized red cell cultures as well as in uninfected cells kept in in vitro, as compared with normal erythrocytes. The consequence of such changes induced by the malarial parasites in the host red cells in the pathogenesis of erythrocyte destruction and anemia of P. falciparum malaria is discussed.


Platelet heterogeneity based on lactic dehydrogenase activity. Mean lactic dehydrogenase (LDH) activity of human platelets is about 5 nU/platelet; expressed on a cell volume basis, it is 0.5 nU/fl. Platelet separation by means of centrifugation on a Percoll discontinuous gradient gives different subpopulations with a clear-cut change of LDH at a mean platelet volume (MPV) of about 6 fl. The LDH content of platelets with a MPV > 6 fl is constant (about 0.5 nU/fl), while that of small platelets (3 < MPV < 6 fl) is inversely correlated with the MPV and reaches a value of 1.5-2.0 nU/fl. Because small platelets are granule-depleted, we suggest that platelet LDH activity be considered as an in vivo activation index.


Histoplasmosis in hairy cell leukemia: case report and review of the literature. Progressive disseminated histoplasmosis (PDH) has been described in only six patients with hairy cell leukemia (HCL). Herein we describe an additional patient with HCL and disseminated histoplasmosis. Additionally, we note that three of seven cases of disseminated histoplasmosis and HCL have occurred in East Texas. PDH is to be suspected in febrile HCL patients in an endemic area who fail to respond to antibacterial therapy. We emphasize that serologic studies are useful in the diagnosis of PDH in HCL patients, and these patients respond well to therapy.


Secondary acute lymphoblastic leukemia with t (4;11): report on two cases and review of the literature. We report two cases of secondary acute lymphoblastic leukemia (ALL) with t (4;11) (q21;q23) translocation occurring after chemotherapy and radiotherapy for a prior cancer. Seven previously published cases of secondary ALL with t (4;11) (q21;q23) are also reviewed. Most patients had received a combination of topoisomerase II inhibitors (anthracyclines, mitoxantrone, or the epipodophillotoxin derivatives VP16 or VM26) and cyclophosphamide, which have also been implicated in the pathogenesis of secondary acute myeloid leukemia (AML) with 11q23 rearrangements. These observations give further support to the existence of a subgroup of secondary acute leukemias with cytogenetic findings "specific" for de novo ALL and AML, especially those with translocations involving the 11q23 region.


Health and demographic characteristics of twin births: United States, 1988. National trends in twin birth incidence by race of child are analyzed for the period 1950-88. Also reviewed are maternal and infant health and demographic characteristics associated with twin delivery for the year 1988.


Mortality pattern of German vegetarians after 11 years of follow-up. A cohort of 1,904 vegetarians and persons leading a health-conscious life-style in the Federal Republic of Germany was identified in 1978. After a follow-up of 11 years, mortality from all causes was reduced by one-half compared with the general population [the standardized mortality ratio (SMR) was 0.44 for men, 0.53 for women]. Among the 858 men, 111 deaths were observed, with 255 expected; among the 1,046 women, 114 deaths were observed, with 215 expected. The lowest mortality was found for cardiovascular diseases (SMR = 0.39 for men, 0.46 for women); in particular, for ischemic heart diseases, mortality was reduced to one-third of that expected. Cancer mortality was reduced by one-half in men (SMR = 0.48), but only by one-quarter in women (SMR = 0.74). The deficit in cancer deaths was mainly observed for lung cancer and gastrointestinal cancers in males and for gastrointestinal cancers in females. Deaths from diseases of the respiratory and digestive systems were also reduced by about 50%. An excess of deaths occurred only for anemia. When the strict and the moderate vegetarians were analyzed separately, the strongest differential was found for ischemic heart diseases, which were much less frequent among strict vegetarians for both sexes. Some nondietary factors, such as higher socioeconomic status, virtual absence of smoking, and lower body mass index, may also have contributed to the lower mortality of the study participants.


Dietary trace elements and esophageal cancer mortality in Shanxi, China. To explore the relation between esophageal cancer and dietary trace elements in humans, we estimated the average daily intake of zinc, copper, iron, selenium, molybdenum, silicon, cadmium, and nickel in 21 Chinese communes, where the annual mortality rate from esophageal cancer among the population 30 years of age and over ranged from 0 to 495/100,000 person-years. We also estimated the relative level of calcium consumption. Zinc and copper intake were inversely related to esophageal cancer mortality, and calcium intake levels was positively related to esophageal cancer mortality. The predicted esophageal cancer mortality among a vegetarian population with a high level of dietary calcium and a low level of dietary zinc was 5.3 times as high as that in a vegetarian population with a low level of dietary calcium and a high level of dietary zinc. The influence of a high level of dietary calcium in a vegetarian population may be explained by a reduction in the absorption of dietary zinc.


The association of waterborne chloroform with intrauterine growth retardation. The potential reproductive effects of long-term, low-dose exposure to chloroform have received little attention despite the known, acute toxicity of high exposures and the wide-spread occurrence of low concentrations in drinking water. We studied the association of waterborne chloroform with low birthweight (less than 2,500 gm), prematurity (less than 37 weeks gestation), and intrauterine growth retardation (less than 5th percentile of weight for gestational age). Cases were not mutually exclusive, but each outcome was analyzed independently. Birth certificates from January 1, 1989, to June 30, 1990, were used to identify cases and randomly selected controls. All were live, singleton infants born to non-Hispanic, white women from Iowa towns with 1,000-5,000 inhabitants. Exposures to chloroform and other trihalomethanes were ecologic variables based on maternal residence and a 1987 municipal water survey. After adjustment for maternal age, parity, adequacy of prenatal care, marital status, education, and maternal smoking by multiple logistic regression, residence in municipalities where chloroform concentrations were greater than or equal to 10 micrograms/liter was associated with an increased risk for intrauterine growth retardation (odds ratio = 1.8, 95% confidence interval = 1.1-2.9). The major limitations of this study involve the ascertainment and classification of exposures to trihalomethanes, including such issues as the imprecision of using aggregate municipal measures for classifying exposure at the level of the individual, the potential misclassification due to residential mobility, and the fluctuation of trihalomethane levels.


Oral contraceptives and premenopausal bilateral breast cancer: a case-control study. We estimated the effect of oral contraceptive (OC) use on premenopausal bilateral breast cancer in a matched case-control study. One hundred forty-four cases were identified from population-based registries of Los Angeles County, California, and of Connecticut and from the major hospitals in Montreal and Quebec City. Matched controls were the unaffected sisters of the cases. When age was included in the model, ever-use of OCs for 1 year or more was associated with an odds ratio 1.7 (95% confidence interval = 1.0-2.9). The odds ratios associated with 1-2, 3-6, and 7 years of use were 1.2 (0.61-2.4), 2.5 (1.2-5.3), and 2.0 (0.93-4.2), respectively. Too few women had used OCs before their first full-term pregnancy or before age 25 for these estimates to be informative. Restricting the analyses to women who had ever given birth yielded an odds ratio for ever-use of OCs of 2.1 (1.0-4.4). The results indicate an increased risk of premenopausal bilateral breast cancer associated with OC use.


Notes on the assessment of trend in the presence of nondifferential exposure misclassification. Estimation of overall linear trend and statistical tests for trend are commonly applied in epidemiologic studies to evaluate the association between ordinal exposure variables and dichotomous health outcomes. In the discussion of these studies, the assertion is commonly made that the observed trends can only be conservative and reported P-values can only be too high owing to the occurrence of exposure misclassification that is supposed to be nondifferential, that is, unrelated to the health outcome of interest. This paper illustrates that this assertion is not generally justified without additional sensitivity analyses. Overestimation of trends and an erroneous reduction of P-values due to nondifferential exposure misclassification are unlikely, but not impossible, for monotonic exposure-disease associations. These may be common occurrences, however, if the observed exposure-disease association is nonmonotonic.


Preterm birth subtypes among blacks and whites. The differences in preterm birth between blacks and whites are poorly understood. Our study examined subtypes of moderately preterm delivery (34-36 completed weeks of gestation) and very preterm delivery (20-33 weeks) in blacks and whites using North Carolina birth certificate data for 1988-1989. We divided the causes of preterm birth into three categories: preterm premature rupture of the membranes, indication of pregnancy complication, and idiopathic preterm deliveries. The overall prevalence of preterm birth was 8.0% and 16.7% for whites and blacks, respectively. The entire gestational age distribution of blacks was shifted to earlier ages relative to whites. More highly educated blacks still had higher risks of moderately and very preterm deliveries than less educated whites. Multivariate analysis, controlling for other factors, showed that blacks had 3.3, 2.5, and 3.5 times the risk of whites to have preterm premature rupture of the membranes, complication-related, and idiopathic delivery, respectively, among very preterm births, and 1.6, 1.9, and 2.0 times the risk of whites for moderately preterm births of the same three types.


Dichotomizing continuous outcome variables: dependence of the magnitude of association and statistical power on the cutpoint. Dichotomizing a continuous outcome variable casts that variable in traditional epidemiologic terms (that is, disease, no disease). One consequence is overall reduced statistical power. A more fundamental concern is that the magnitude of various measures of association (for example, prevalence ratio, odds ratio) and statistical power depend on the cutpoint used to dichotomize the variable. The phenomenon is illustrated with a hypothetical situation assuming a two-level predictor variable and a normally distributed outcome variable. As the cutpoint is increased from lower to higher values, the prevalence ratio increases steadily, the odds ratio is described by a U-shaped curve, and statistical power is described by an inverted U-shaped curve. Furthermore, the extent of these effects depends on the difference between the means of the continuous outcome variable for the two levels of the predictor variable. An empirical example is given using data on education and blood pressure (dichotomized to create a high blood pressure vs low blood pressure variable). Except at each end of the distribution, the results follow the hypothetical example. The observation has implications for public health and medical treatment; different cutpoints should be examined to determine the optimal cutpoint in terms of policy and/or treatment decisions. The observation described here also has implications for statistical interpretation; statements about the magnitude of association or statistical significance have limited meaning unless both the cutpoint and the distribution of the outcome variable are specified.