Cardiogenic shock: A look at acute functional mitral incompetence.

A 44-year-old man presented with cardiogenic shock secondary to acute functional mitral incompetence as well as septic shock related to pneumonia. The patient deteriorated haemodynamically despite adequate medical therapy. An echocardiogram revealed a massive mitral incompetence and an ejection fraction of 32%. An intra-aortic balloon pump was placed and the patient improved dramatically. On day 6 after admission the echocardiogram was repeated, revealing a mild mitral incompetence and an ejection fraction of 58%.

Yeasts Associated with Culex pipiens and Culex theileri Mosquito Larvae and the Effect of Selected Yeast Strains on the Ontogeny of Culex pipiens.

The success of mosquitoes in nature has been linked to their microbiota and bacteria in particular. Yet, knowledge on their symbioses with yeasts is lacking. To explore possible associations, culturable yeasts were isolated from wild larvae of Culex pipiens and Culex theileri. These yeasts were classified using restriction fragment length polymorphism (RFLP) analyses and identified by sequencing the D1/D2 region of the 26S rRNA gene. Representative strains of Candida, Cryptococcus, Galactomyces, Hannaella, Meyerozyma, Pichia, Rhodosporidium, Rhodotorula, Trichosporon and Wickerhamomyces were isolated. Our results provide, to our knowledge, the first records of the yeast microbiota from wild mosquito larvae and show that they may harbour potential clinically relevant yeast species, including the well-known opportunistic human pathogen Candida albicans. Also, diminished numbers of yeast isolates originating from adults, compared to larvae, support the hypothesis of microbial reduction/elimination during adult emergence and extend it to include yeasts. In addition, strains of Candida albicans, Candida glabrata, Candida pseudolambica, Cryptococcus gattii, Metschnikowia bicuspidata, Saccharomyces cerevisiae and Wickerhamomyces anomalus were tested as sole feed during a 21-day feeding experiment wherein cumulative larval growth, survival and pupation of Cx. pipiens were recorded. Although most yeasts supported larval growth in a similar manner to the positive control S. cerevisiae strain, the different yeast strains impacted differently on Culex pipiens ontogeny. Notably, survival and pupation of larvae were negatively impacted by a representative strain of the primary pathogen C. gattii - signifying some yeasts to be natural antagonists of mosquitoes.

Characterization of the Mycobacterium tuberculosis iniBAC promoter, a promoter that responds to cell wall biosynthesis inhibition.

The cell wall provides an attractive target for antibiotics against Mycobacterium tuberculosis. Agents such as isoniazid and ethambutol that work by inhibiting cell wall biosynthesis are among the most highly effective antibiotics against this pathogen. Although considerable progress has been made identifying the targets for cell wall active antibiotics, little is known about the intracellular mechanisms that are activated as a consequence of cell wall injury. These mechanisms are likely to have an important role in growth regulation and in the induction of cell death by antibiotics. We previously discovered three isoniazid-induced genes (iniB, iniA, and iniC) organized in tandem on the M. tuberculosis genome. Here, we investigate the unique features of the putative iniBAC promoter. This promoter was specifically induced by a broad range of inhibitors of cell wall biosynthesis but was not inducible by other conditions that are toxic to mycobacteria via other mechanisms. Induction required inhibitory concentrations of antibiotics and could be detected only in actively growing cells. Analysis of the iniBAC promoter sequence revealed both a regulatory element upstream and a potential repressor binding region downstream of the transcriptional start site. The induction phenotype and structure of the iniBAC promoter suggest that a complex intracellular response occurs when cell wall biosynthesis is inhibited in M. tuberculosis and other mycobacteria.

Recent developments in mycobacterial research.

Tuberculosis remains a major health problem in the world, which is compounded further by the alarmingly high rate of M. tuberculosis infections in AIDS patients. Thus, there is an urgent need to advance our understanding of the mycobacterium to develop new drugs. The extraordinary recent developments in mycobacterial genetic research, particularly in genomics will greatly facilitate this goal. The knowledge of the entire genome sequence of M. tuberculosis will help in designing new chemotherapeutic and immunotherapeutic interventions. This review highlights recent developments in genomics, mycobacterial genetics, novel vaccine strategies, and our understanding of tuberculous dormancy.

Cloning, mapping and characterization of a genomic copy of the Lipomyces kononenkoae alpha-amylase-encoding gene (LKA1).

The expression in Saccharomyces cerevisiae and Schizosaccharomyces pombe of a cDNA copy of the Lipomyces kononenkoae IGC4052B alpha-amylase gene (LKA1), linked to the phosphoglycerate kinase gene (PGK1) promoter, resulted in the extracellular production of biologically active alpha-amylase (LKA1). However, transformation of S. cerevisiae and Schiz. pombe with a cosmid clone containing the complete genomic copy of LKA1, expressed from its native promoter, did not result in secretion of active alpha-amylase by any of the transformants. When the cDNA copy of LKA1 was expressed in S. cerevisiae under control of the wild-type L, kononenkoae promoter, biologically active alpha-amylase was secreted into the culture medium, indicating the recognition of the LKA1 promoter in S. cerevisiae. Sequence analysis of the GC-rich LKA1 promoter revealed canonical sequences that are homologous to the TATAAA, CAAT and CCAAT boxes and GCN4-binding sites that are present in several promoter sequences of S. cerevisiae. Primer extension analysis of LKA1 transcripts in L. kononenkoae indicated major initiation sites at nucleotides -64 and -65. S. cerevisiae and Schiz. pombe cells transformed with a plasmid containing the open reading frame of the genomic copy of LKA1, linked to the PGK1 promoter, did not produce alpha-amylase. Polymerase chain reaction mapping and sequence analysis revealed the presence of a 61-bp intron in the genomic copy of LKA1 that impaired synthesis of biologically active alpha-amylase in S. cerevisiae and Schiz. pombe. This intron contains donor, acceptor and branch sequences that correlate with the consensus sequences identified in the introns of split genes from Schiz. pombe and mammals. Pulsed-field gradient gel electrophoresis resolved at least eight chromosomal DNAs for L. kononenkoae IGC4052B and chromoblot analysis indicated that LKA1 is located on the second smallest chromosome, designated chromosome II.

Cloning, sequence analysis and expression in yeasts of a cDNA containing a Lipomyces kononenkoae alpha-amylase-encoding gene.

The yeast Lipomyces kononenkoae (Lk) secretes a highly active raw starch-degrading alpha-amylase (alpha Amy) that liberates reducing groups from glucose polymers containing both alpha-1,4 and alpha-1,6 bonds. The LKA1 gene encoding this industrially important alpha Amy was cloned as a 2261-bp cDNA fragment from a glucose-derepressed mutant (IGC4052B) of Lk and characterized. The nucleotide (nt) sequence of the cDNA fragment was determined, revealing an open reading frame of 1872 bp, encoding a 596 amino-acid (aa) mature protein (LKA1) with a calculated M(r) of 65,706. The similarity between the aa sequence of LKA1 and those of other alpha Amy showed four common conserved regions characteristic of the alpha Amy protein family: (A) 264DIVVNH269, (B) 349GLRIDTVKH357, (B') 376GEVFD380 and (C) 439FLENQD444. The deduced aa sequence revealed significant homology to the aa sequences of the Aspergillus oryzae, Schwanniomyces occidentalis and Saccharomycopsis fibuligera alpha Amy, various bacterial cyclomaltodextrin glucanotransferases, a beta-amylase and the 5'-region of a glucoamylase. LKA1 was expressed in Saccharomyces cerevisiae (Sc) under the control of the phosphoglycerate kinase (PGK1) promoter and Northern blot analysis showed the presence of a single 2.3-kb transcript. The 28-aa signal peptide of the LKA1 protein efficiently directed its secretion into the medium when expressed in Sc.

Characterization of a novel alpha-amylase from Lipomyces kononenkoae and expression of its gene (LKA1) in Saccharomyces cerevisiae.

A highly active alpha-amylase (76,250 Da) secreted by the raw starch-degrading yeast Lipomyces kononenkoae strain IGC4052B was purified and characterized. Using high performance liquid chromatography (HPLC), end-product analysis indicated that the L. kononenkoae alpha-amylase acted by endo-hydrolysis on glucose polymers containing alpha-1,4 and alpha-1,6 bonds, producing mainly maltose, maltotriose and maltotetraose. The following NH2-terminal amino acids were determined for the purified enzyme: Asp-Cys-Thr-Thr-Val-Thr-Val-Leu-Ser-Ser-Pro- Glu-Ser-Val-Thr-Gly. The L. kononenkoae alpha-amylase-encoding gene (LKA1), previously cloned as a cDNA fragment, was expressed in Saccharomyces cerevisiae under the control of the PGK1 promoter. The native signal sequence efficiently directed the secretion of the glycosylated protein in S. cerevisiae. De-glycosylation of the enzyme indicated that post-translational glycosylation is different in S. cerevisiae from that in L. kononenkoae. Zymogram analysis indicated that glycosylation of the protein in S. cerevisiae had a negative effect on enzyme activity. Southern-blot analysis revealed that there is only a single LKA1 gene present in the genome of L. kononenkoae.

Coronary heart disease risk factors in a rural and urban Orange Free State black population.

OBJECTIVE: To determine and compare the prevalence of ischaemic heart disease (IHD) risk factors in a rural and an urban black population. DESIGN: A survey to determine the prevalence of hypertension, diabetes mellitus, smoking, obesity, central obesity and dyslipidaemia in black subjects 25 years and older. SETTING: The indigenous black populations of QwaQwa and Mangaung. PARTICIPANTS: A random sample of 950 households was selected from each area. From each household an unrelated male and/or female subject was selected in a standardised way. From QwaQwa 853 subjects (279 men and 574 women) and from Mangaung 758 subjects (290 men and 468 women) participated in the study. The response rate was 68% and 62% respectively for QwaQwa and Mangaung. MAIN OUTCOME MEASURES: Few urban-rural differences in the prevalence of IHD risk factors were found in this study. A low prevalence of clustering of major IHD risk factors was noted. RESULTS: The age- and sex-adjusted prevalences of hypertension were 29% in QwaQwa and 30.3% in Mangaung. Diabetes was present in 4.8% of the QwaQwa sample and 6% of the Mangaung sample. The prevalence of heavy smoking in the Mangaung sample was almost double that of the QwaQwa sample and mostly confined to men. High-risk hypercholesterolaemia was present in 12.5% of QwaQwa and 6% of Mangaung men in the 25-34-year age group. The corresponding figures for moderate-risk hypercholesterolaemia were 34% and 44.8% and both levels of risk declined with increasing age. The mean body mass index of women in both samples exceeded 25 kg/m2. CONCLUSION: All the elements for a potential epidemic of atherosclerotic cardiovascular disease are present in the study populations. The similarity of findings in the two samples may be indicative of the advanced stage of urbanisation and westernisation of the rural group. It is alarming that subjects in the younger age groups tended to have the highest prevalences of moderate and even high-risk hypercholesterolaemia.

Expression of human P450c17 as an export protein in Saccharomyces cerevisiae.

Cytochrome P450c17 (P450c17), together with cytochrome P450c21 (P450c21), plays an important role in progesterone metabolism in the mammalian adrenal cortex. Low levels of expression and the presence of other steroidogenic enzymes in adrenal cortex endoplasmic reticulum (ER) impedes purification and characterisation of wild type as well as mutant forms of the hemoprotein. Heterologous gene expression systems have previously been used successfully to express active P450c17. Heterologous expression can also be used for the preparation of anti-P450c17-IgG. For antibody production larger amounts of pure P450c17 peptide, rather than the active protein, is, however, desirable. If the expressed protein can be affinity tagged and secreted into the medium, isolation and purification will be facilitated. Saccharomyces cerevisiae, YPH259, was transformed with a modified YCplac111 yeast expression-secretion vector (pPRL2). The gene coding for a truncated human P450c17 (signal anchor sequence 1-18 was removed) was inserted, in reading frame, downstream from the leader sequence MF alpha. A histidine tag was incorporated at the C-terminus. The modified yeast expression vector was expressed in yeast, the secreted P450c17-peptide purified by affinity chromatography and identified by immunoblot analysis.

Regional sequence homologies in starch-degrading enzymes.

The enzymatic hydrolysis of starch, consisting of linear (amylose) and branched (amylopectin) glucose polymers, is catalyzed by alpha-, beta- and glucoamylases (gamma-amylases), cyclodextrinases, alpha-glucosidases, and debranching enzymes. Saccharomyces cerevisiae cannot utilize starch. Our laboratory has previously co-expressed the Bacillus amyloliquefaciens alpha-amylase (AMY) and the Saccharomyces diastaticus glucoamylase (STA2) genes in S. cerevisiae. A gene encoding a debranching enzyme (pullulanase) from Klebsiella pneumoniae ATCC15050 was cloned and its nucleotide sequence determined. This gene will be co-expressed with the alpha- and gamma-amylase to produce an amylolytic S. cerevisiae strain. Extensive data base comparisons of the K. pneumoniae pullulanase amino-acid sequence with the amino-acid sequences of other debranching enzymes and alpha-, beta- and gamma-amylases (from bacteria, yeasts, higher fungi and higher eukaryotes), indicated that these debranching enzymes have amino-acid regions similar to those found in alpha-amylases. The conserved regions in alpha-amylases comprise key residues that are implicated in substrate binding, catalysis, and calcium binding and are as follows. Region 1: DVVINH; region 2: GFRLDAAKH and region 4: FVDNHD. When comparing conserved regions, no similarity could be detected between debranching enzymes and beta- and gamma-amylases.

Expression and secretion of Bacillus amyloliquefaciens alpha-amylase by using the yeast pheromone alpha-factor promoter and leader sequence in Saccharomyces cerevisiae.

Replacement of the regulatory and secretory signals of the alpha-amylase gene (AMY) from Bacillus amylolique-faciens with the complete yeast pheromone alpha-factor prepro region (MF alpha 1p) resulted in increased levels of extracellular alpha-amylase production in Saccharomyces cerevisiae. However, the removal of the (Glu-Ala)2 peptide from the MF alpha 1 spacer region (Lys-Arg-Glu-Ala-Glu-Ala) yielded decreased levels of extracellular alpha-amylase.

Co-expression of a Saccharomyces diastaticus glucoamylase-encoding gene and a Bacillus amyloliquefaciens alpha-amylase-encoding gene in Saccharomyces cerevisiae.

A glucoamylase-encoding gene (STA2) from Saccharomyces diastaticus and an alpha-amylase-encoding gene (AMY) from Bacillus amyloliquefaciens were cloned separately into a yeast-integrating shuttle vector (YIp5), generating recombinant plasmids pSP1 and pSP2, respectively. The STA2 and AMY genes were jointly cloned into YIp5, generating plasmid pSP3. Subsequently, the dominant selectable marker APH1, encoding resistance to Geneticin G418 (GtR), was cloned into pSP3, resulting in pSP4. For enhanced expression of GtR, the APH1 gene was fused to the GAL10 promoter and terminated by the URA3 terminator, resulting in pSP5. Plasmid pSP5 was converted to a circular minichromosome (pSP6) by the addition of the ARS1 and CEN4 sequences. Laboratory strains of Saccharomyces cerevisiae transformed with plasmids pSP1 through pSP6, stably produced and secreted glucoamylase and/or alpha-amylase. Brewers' and distillers' yeast transformed with pSP6 were also capable of secreting amylolytic enzymes. Yeast transformants containing pSP1, pSP2 and pSP3 assimilated soluble starch with an efficiency of 69%, 84% and 93%, respectively. The major starch hydrolysis products produced by crude amylolytic enzymes found in the culture broths of the pSP1-, pSP2- and pSP3-containing transformants, were glucose, glucose and maltose (1:1), and glucose and maltose (3:1), respectively. These results confirmed that co-expression of the STA2 and AMY genes synergistically enhanced starch degradation.

Diabetes mellitus, pulmonary tuberculosis and chronic calcific pancreatitis revisited.

The prevalence of chronic calcific pancreatitis (CCP) was determined in 25 successive patients with both diabetes mellitus and newly diagnosed pulmonary tuberculosis. Twenty patients (80%) were alcoholics and all were black. Of these, 9 (45%) had CCP. In only 3 of these 9 patients was the history compatible with the condition diagnosed. Clinical steatorrhoea was absent in the patients with CCP. Pulmonary tuberculosis was extensive with major involvement of three or more lung zones in 36% of patients. Mainly basal involvement of the lungs was present in 8% of patients.

Ophthalmoscopy versus non-mydriatic fundus photography in the detection of diabetic retinopathy in black patients.

The contribution of non-mydriatic fundus photography in the detection of diabetic retinopathy before and after dilatation of the pupils in black diabetics was investigated and compared with direct ophthalmoscopy. Eighty-six patients were examined and good-quality photographs were obtained for 54.7% of eyes before and 86.6% of eyes after dilatation. Photographically documented retinopathy was detected by ophthalmoscopy in only 64.7% of eyes. The two methods were concordant for the presence of retinopathy in 62.2% of eyes before and 56.9% of eyes after dilatation. Photography through dilated pupils also improved the rate of detection of diabetic retinopathy from 24% to 30%. The 45 degrees non-mydriatic fundus camera was found to be a valuable adjunct in the detection of diabetic retinopathy in a busy diabetic clinic.

A physical analysis of the prophage of Proteus mirabilis PM5006.

Proteus mirabilis phage 5006M was investigated as a prophage by DNA hybridization. A physical map of the prophage was established and the site of integration was localized on the phage genome. Older reports that the phage is cryptic in P. mirabilis could not be verified.

Autonomic neuropathy and atypical myocardial infarction in a diabetic clinic population.

The incidence of autonomic neuropathy in 52 insulin-dependent and 87 non-insulin-dependent diabetic subjects was studied as well as the relationship between the type and duration of diabetes, metabolic control and the association of atypical infarctions with autonomic neuropathy. No statistically significant relationship was found between any parameter and the presence of autonomic neuropathy. Atypical infarctions were not confined to subjects with autonomic neuropathy, but the incidence was considerably higher than that in the general population. Position of the infarction did not determine whether it was silent or not. It is suggested that physicians should be alert to the occurrence of silent infarctions in all diabetics and not only those with clinical evidence of autonomic neuropathy.

Evaluation of gastrointestinal motility using the hydrogen breath test.

The purpose of the study was to evaluate the validity of a model where intestinal transit is increased and decreased by motility modifying drugs. The measurement of breath hydrogen concentrations after ingestion of lactulose was used to estimate small intestinal transit time. After obtaining base-line values, eight healthy volunteers were pretreated on separate occasions with loperamide, diphenoxylate, metoclopramide and cisapride. Diphenoxylate caused a significant increase in small bowel transit time, whereas both metoclopramide and cisapride significantly shortened it. The H2 breath test therefore seems to accurately reflect the expected transit time. Loperamide did not alter significantly intestinal transit. Possibly this drug counteracts its own delaying influence on small bowel transit by hurrying gastric emptying. Alternatively, not enough time was allowed for it to exert its full effect.

Systemic nodular panniculitis (Weber-Christian disease) in a black woman. A case report.

There are many causes of panniculitis and systemic nodular fat necrosis; Weber-Christian disease is one of the rarer forms. A 47-year-old black woman with Weber-Christian disease is described and the aetiology, pathology and clinical manifestations of the disease are discussed.

Ultrasonography in the localization of parathyroid adenomas.

The use of ultrasonography in the diagnosis of a parathyroid adenoma is described, and the limitations and advantages of this method are discussed.