Comparison of paediatric infectious disease deaths in public sector health facilities using different data sources in the Western Cape, South Africa (2007-2021).

BACKGROUND: Routinely collected population-wide health data are often used to understand mortality trends including child mortality, as these data are often available more readily or quickly and for lower geographic levels than population-wide mortality data. However, understanding the completeness and accuracy of routine health data sources is essential for their appropriate interpretation and use. This study aims to assess the accuracy of diagnostic coding for public sector in-facility childhood (age < 5 years) infectious disease deaths (lower respiratory tract infections [LRTI], diarrhoea, meningitis, and tuberculous meningitis [TBM]) in routine hospital information systems (RHIS) through comparison with causes of death identified in a child death audit system (Child Healthcare Problem Identification Programme [Child PIP]) and the vital registration system (Death Notification [DN] Surveillance) in the Western Cape, South Africa and to calculate admission mortality rates (number of deaths in admitted patients per 1000 live births) using the best available data from all sources. METHODS: The three data sources: RHIS, Child PIP, and DN Surveillance are integrated and linked by the Western Cape Provincial Health Data Centre using a unique patient identifier. We calculated the deduplicated total number of infectious disease deaths and estimated admission mortality rates using all three data sources. We determined the completeness of Child PIP and DN Surveillance in identifying deaths recorded in RHIS and the level of agreement for causes of death between data sources. RESULTS: Completeness of recorded in-facility infectious disease deaths in Child PIP (23/05/2007-08/02/2021) and DN Surveillance (2010-2013) was 70% and 69% respectively. The greatest agreement in infectious causes of death were for diarrhoea and LRTI: 92% and 84% respectively between RHIS and Child PIP, and 98% and 83% respectively between RHIS and DN Surveillance. In-facility infectious disease admission mortality rates decreased significantly for the province: 1.60 (95% CI: 1.37-1.85) to 0.73 (95% CI: 0.56-0.93) deaths per 1000 live births from 2007 to 2020. CONCLUSION: RHIS had accurate causes of death amongst children dying from infectious diseases, particularly for diarrhoea and LRTI, with declining in-facility admission mortality rates over time. We recommend integrating data sources to ensure the most accurate assessment of child deaths.

Laboratory parameters associated with prolonged hospital length of stay in COVID-19 patients in Johannesburg, South Africa.

BACKGROUND: Coronavirus disease (COVID-19) has imposed unprecedented stressors on South Africa (SA)'s healthcare system. Superimposed on the country's quadruple burden of disease, pandemic-related care further exposes existing inequities. Some of these inequities are specific to hospital-based inpatient services, such as the geographical maldistribution of hospital beds, lack of oxygen supplies and assisted ventilation, and scarcity of trained healthcare workers. Certain high-risk groups, such as individuals with cardiometabolic comorbidity, are likely to develop severe COVID-19 disease requiring hospitalisation with potential for a prolonged length of stay (LoS). It may be helpful for health authorities to identify those at risk for prolonged LoS to facilitate appropriate health systems planning. OBJECTIVES: To identify hospital admission laboratory parameters associated with a hospital stay >14 days in patients with COVID-19 pneumonia. METHODS: A retrospective observational study design was used. Laboratory data were obtained from an SA private laboratory for 642 inpatients with suspected or confirmed COVID-19 pneumonia, comprising 7 months of admission laboratory data from six private hospitals in Johannesburg, Gauteng Province. RESULTS: Of 642 hospital admissions for pneumonia, 497 were confirmed to have COVID-19 infection (reverse transcription-polymerase chain reaction test positive). In the COVID-19-positive group, hospital LoS was prolonged in 35.4% of admissions. Univariate analysis demonstrated an association with the following risk factors for prolonged LoS: older age; male sex; high serum creatinine, sodium (Na), chloride, potassium and urea levels and low estimated glomerular filtration rate; raised white blood cell count, lymphopenia, neutrophilia and an elevated neutrophil-to-lymphocyte ratio (NLR); and elevated levels of D-dimers, interleukin-6 (IL-6), and procalcitonin (PCT). The strongest univariate associations (relative risk (RR) ≥2.0) with a hospital stay >14 days were high Na levels, NRL >18, high PCT levels and IL-6 >40 pg/mL. On multivariable analysis, the following factors remained significantly associated with prolonged LoS: older age (RR 1.015 per year of age; 95% confidence interval (CI) 1.005 - 1.024); hypernatraemia (RR 1.80; 95% CI 1.25 - 2.60); hyperkalaemia (RR 1.61; 95% CI 1.18 - 2.20); and neutrophilia (RR 1.47; 95% CI 1.15 - 1.88). CONCLUSIONS: COVID-19 pandemic preparedness requires hospital-based inpatient care to be prioritised in resource-limited settings, and availability of beds and prompt admissions are essential to ensure good clinical outcomes. In this study of COVID-19 patients admitted with pneumonia, multivariable analysis showed older age, hypernatraemia, hyperkalaemia and neutrophilia to be associated with LoS >14 days. This may assist with healthcare systems planning.

Safety of long-term isoniazid preventive therapy in children with HIV: a comparison of two dosing schedules.

SETTING: Two paediatric hospitals in Cape Town, South Africa. OBJECTIVE: To investigate the incidence of and risk factors for severe liver injury in human immunodeficiency virus (HIV) infected children receiving long-term isoniazid preventive therapy (IPT). DESIGN: Randomised trial of IPT or placebo given daily or thrice weekly to HIV-infected children aged ≥8 weeks; placebo was discontinued early. Alanine transaminase (ALT) was measured at baseline, 6-monthly and during illness: an increase of ≥10 times the upper limit of normal defined severe liver injury. RESULTS: Of 324 children enrolled, 297 (91.6%) received IPT (559.1 person-years [py]). Baseline median age was 23 months (interquartile range [IQR] 9.5-48.6) and median CD4%, 20% (IQR 13.6-26.9). A total of 207 (63.9%) children received combination antiretroviral therapy: 19 developed severe liver injury, 16 while receiving IPT. Among these there were 8 cases of viral hepatitis (5 with hepatitis A), 2 antiretroviral-induced liver injuries and 1 case of abdominal tuberculosis. IPT-related severe liver injury occurred in 1.7% (5/297, 0.78/100 py). No child developed hepatic failure; one died of an unrelated cause. All surviving children subsequently tolerated IPT. CONCLUSIONS: This study suggests that long-term IPT has a low toxicity risk in HIV-infected children. In the absence of chronic viral hepatitis, IPT can be safely re-introduced following recovery from liver injury.

A novel CYBB mutation with the first genetically confirmed case of chronic granulomatous disease in South Africa.

A case of a child with chronic granulomatous disease (CGD) presenting with recurrent mycobacterial infections and invasive Aspergillus fumigatus disease is described. Genetic analysis confirmed X-linked CGD with a novel mutation in exon 10 of the CYBB gene - the first South African report of genetically confirmed CGD.

Cerebral monitoring during carotid endarterectomy--a comparison between electroencephalography, transcranial cerebral oximetry and carotid stump pressure.

OBJECTIVE: Various modalities are used for cerebral monitoring during carotid endarterectomy (CEA). The aim of this study was to evaluate whether transcranial cerebral oximetry (TCO) and carotid stump pressure (SP) are as accurate as electroencephalography (EEG) for monitoring cerebral ischaemia during carotid cross-clamping. METHODS: One hundred consecutive patients who underwent CEA were studied with continuous and simultaneous EEG and TCO. SP was measured for each patient. The percentage decrease of oxygenation on TCO was calculated during cross-clamping and surgery. EEG findings were used as the benchmark to detect cerebral ischaemia and were the indication for insertion of a temporary shunt. The relationship with TCO was observed in terms of percentage decrease in oxygenation. RESULTS: A total of 6 patients were shunted on the basis of their EEG changes. TCO changed more than 20% in these 6 patients, but an additional 12 patients had TCO changes with a normal EEG. This correlated with a decrease in blood pressure (BP) and was corrected by increasing the BP. The positive predictive values (PPVs) and negative predictive values (NPVs) for shunting based on TCO (as compared with EEG) were 33% and 100% respectively. Thirty-four patients had SP <50 mmHg, of whom 4 were shunted based on EEG changes. Two of 66 patients with SP >50 mmHg were shunted based on EEG changes. If a shunting policy had been based on a SP of 50 mmHg, 30 patients would have been shunted unnecessarily (PPV 12%), whereas the non-requirement for a shunt was predicted correctly in 64 of 66 patients (NPV 97%). There were 2 major strokes: 1 contralateral on day 3 in a patient with bilateral severe stenoses, and 1 ipsilateral in a nonshunted patient with normal EEG, TCO and SP >50 mmHg. CONCLUSION: Compared with EEG, TCO is a practical and non-invasive monitoring system with a high sensitivity (100%) but a low specificity. TCO is more sensitive to a drop in BP and responds earlier to these changes than EEG. SP should not be used as the sole predictor for shunting during CEA.

Osteopontin is upregulated by BCR-ABL.

Chronic myelogenous leukemia (CML) is characterized by its hallmark oncogene BCR-ABL and the progression from a chronic phase toward an acute leukemia, with a differentiation arrest of the leukemic clone. In the present study, we conducted a microarray analysis using an inducible model of BCR-ABL expression based on the TET-OFF system, and we found that osteopontin (OPN), a component of stem cell niche, is overexpressed in BCR-ABL-expressing cells. Studies using mutant forms of BCR-ABL demonstrated that the BCR-ABL-induced OPN overexpression was a tyrosine kinase-dependent event. Furthermore, OPN concentration was significantly increased in the serum of leukemic mice generated by transplantation of BCR-ABL-expressing bone marrow cells. Most importantly, a significant increase of OPN concentration was observed in the serum of CML patients as compared to controls. Overall these results show that OPN is deregulated by BCR-ABL oncogene and suggest that OPN could be involved in CML stem cell biology.

Serum p53 antibodies in small cell lung cancer: the lack of prognostic relevance.

Prognostic relevance of serum p53 antibodies was assessed in 96 patients with microscopically proven small cell lung cancer (SCLC). The study group included 67 males and 29 females; mean age 58 years; range 35--86 years; 60 with limited disease (LD), and 36 with extensive disease (ED). The control group consisted of 41 patients with non-malignant diseases. The presence of p53 antibodies was assayed by the immunoenzymatic method (P53 ELISA kit, PharmaCell, France). Antibodies were present in 26 SCLC cases (27%); 15 (25%) in LD and 11 (31%) in ED. Antibodies were also found in one out of 41 control subjects (2%). There was no correlation between the level of antibodies and clinical characteristics of SCLC patients including age, gender and extent of disease. The median follow-up for the entire group was 30 months (range: 11--39 months). By the time of analysis, 78 patients (82%) had deceased. Median survival in SCLC patients with and without antibodies was 42 and 39 weeks, respectively (log rank, P=0.81). These results indicate the lack of clinical relevance of serum p53 antibodies in SCLC.

The dose-range effects of sufentanil added to 0.125% bupivacaine on the quality of patient-controlled epidural analgesia during labor.

UNLABELLED: To determine the minimal sufentanil concentration required to improve the quality of patient-controlled epidural analgesia during labor, we compared the efficacy of a combination of 0.125% bupivacaine with 1:800,000 epinephrine and different concentrations of sufentanil in a double-blinded randomized study. Concentrations were no sufentanil (n = 66), 0.078 microg/mL sufentanil (n = 65), 0.156 microg/mL sufentanil (n = 65), 0.312 microg/mL sufentanil (n = 65), and 0.468 microg/mL sufentanil (n = 67). The patient-controlled epidural analgesia setting was a 12-mL bolus dose and a 25-min lockout interval. Pain was scored at 5-6 cm, 7-8 cm, and full cervical dilation by using a 10-cm visual analog scale. At full cervical dilation, the pain scores were lower in the groups receiving a solution of at least 0.156 microg/mL sufentanil. Few differences were observed when using the larger concentrations, except for increased pruritus intensity. The duration of labor and the mode of delivery were similar in each group. Rescue analgesia, which consisted of 6 mL of 0.25% bupivacaine, was infrequent and comparable between groups. The use of the pump did not differ between groups. Adding a small concentration of sufentanil to 0.125% bupivacaine for patient-controlled epidural analgesia during labor improved the quality of analgesia but did not modify the bupivacaine requirement. Reducing the sufentanil concentrations to 0.156 microg/mL decreased the pruritus intensity without reducing analgesia. IMPLICATIONS: Adding a small concentration of sufentanil to 0.125% bupivacaine for patient-controlled epidural analgesia during labor improved the quality of analgesia but did not modify the bupivacaine requirement. Reducing the sufentanil concentrations to 0.156 microg/mL decreased the pruritus intensity without reducing analgesia.

Thrombasthenic mice generated by replacement of the integrin alpha(IIb) gene: demonstration that transcriptional activation of this megakaryocytic locus precedes lineage commitment.

To analyze the transcriptional activity of the gene encoding the alpha subunit of the platelet integrin alpha(IIb)beta(3) during the hematopoietic differentiation, mice were produced in which the herpes virus thymidine kinase (tk) was introduced in this megakaryocytic specific locus using homologous recombination technology. This provided a convenient manner in which to induce the eradication of particular hematopoietic cells expressing the targeted gene. Results of progenitor cell cultures and long-term bone marrow (BM) assays showed that the growth of a subset of stem cells was reduced in the presence of the antiherpetic drug ganciclovir, demonstrating that the activation of the toxic gene occurs before the commitment to the megakaryocytic lineage. Furthermore the knock-in of the tk gene into the alpha(IIb) locus resulted in the knock-out of the alpha(IIb )gene in homozygous mice. Cultures of BM cells of these animals, combined with ultrastructural analysis, established that the alpha(IIb) glycoprotein is dispensable for lineage commitment and megakaryocytic maturation. Platelets collected from alpha(IIb)-deficient mice failed to bind fibrinogen, to aggregate, and to retract a fibrin clot. Moreover, platelet alpha-granules did not contain fibrinogen. Consistent with these characteristics, the mice displayed bleeding disorders similar to those in humans with Glanzmann thrombasthenia. (Blood. 2000;96:1399-1408)

Patient-controlled epidural analgesia during labor: the effects of the increase in bolus and lockout interval.

UNLABELLED: Most studies use a bolus size of <6 mL of 0.125% bu- pivacaine for patient-controlled epidural analgesia (PCEA) during labor. In this double-blinded, randomized study, we compared the efficacy of a larger bolus injected via a PCEA pump to a conventional PCEA setting. By using a combination of 0.125% bupivacaine with 1:800,000 epinephrine and 0.625 microg/mL sufentanil, the first PCEA setting was typical (4 mL/8 min), whereas the other combined a 12-mL bolus dose and a 25-min lockout interval, i.e., similar maximal hourly dose. Rescue analgesia was provided with 6 mL of 0.25% bupivacaine. Patient satisfaction and pain were scored on verbal and visual analog scales. Data were analyzed from 103 parturients in the 12-mL/25-min group and 100 in the 4-mL/8-min group. In the 12-mL/25-min group, the median pain score on a 0- to 10-cm visual analog scale was lower at 6-cm cervical dilation (1 [range = 0-8] vs 3 [0-8]) and at delivery (1 [0-10] vs 2 [0-10]). Satisfaction was also better (70% vs 38% "excellent" opinions, at 6-cm cervical dilation). Use of the pump (ratio of successful and total demands) was high and similar in both groups. Rescue analgesia was comparable. Doses of analgesics were greater in the 12-mL/25-min group (hourly bupivacaine dose = 13.9 +/- 5.3 [mean+/- SD] vs 9.4 +/- 4.1 mg). No differences were noted between groups for the severity of hypotension, ephedrine requirement, outcome of the delivery, and Apgar scores. IMPLICATIONS: A patient-controlled epidural analgesia setting that allows a parturient to receive an increased analgesic dose improves satisfaction with patient-controlled epidural analgesia during labor.

Chondrosarcoma and myositis ossificans.

OBJECTIVE: To discuss the clinical radiographic findings in a 70-yr-old woman suffering from chondrosarcoma. CLINICAL FEATURES: The patient experienced right SI pain present initially only at night. She later developed morning numbness. An X-ray examination revealed a flocculent calcification in the right buttock region. Computed tomographic scans confirmed the diagnosis. INTERVENTION AND OUTCOME: Initial palliative care continued until surgery was performed to resect the area. CONCLUSION: Chondrosarcoma is a severe disease that must be differentiated from myositis ossificans.

Ultrastructural analysis of bone marrow hematopoiesis in mice transgenic for the thymidine kinase gene driven by the alpha IIb promoter.

Transgenic mice have been generated with expression of the herpes virus thymidine kinase gene directed by a 2.7-kb fragment of the alphaIIb murine promoter of the gene encoding the alphaIIb-subunit of the platelet integrin alphaIIbbeta3 (Tropel et al, Blood 90:2995, 1997). Administration of ganciclovir (GCV) to these mice resulted not only in an acute cessation of platelet production due to the depletion of the megakaryocytic lineage, but also a decrease in erythrocyte and leukocyte numbers. Immunogold staining on ultrathin frozen sections and electron microscopy has now shown that the remaining population of immature hematopoietic cells contain a high proportion of Sca-1(+) and CD34(+) cells, with CD45R+ cells of the lymphopoietic lineage being maintained. Stromal cells were also preserved. Blood thrombopoietin levels were high. At 4 days of the recovery phase, Sca-1 and CD34 antigen expression decreased with intense proliferation of cells of the three lineages, with megakaryocyte (MK) progenitors being identified by their positivity for glycoprotein IIb-IIIa. These results suggest that transcriptional activity for the alphaIIb gene promoter was present on pluripotent hematopoietic stem cells. At 6 to 8 days after cessation of GCV, numerous mature MK were observed, some of them with deformed shapes crossing the endothelial barrier through thin apertures. Proplatelet production was visualized in the vascular sinus. After 15 days, circulating platelet levels had increased to approximately 65% of normal. Transgenic alphaIIb-tk mice constitute a valuable model to study in vivo megakaryocytopoiesis.

Analysis of hematopoietic stem cell reprogramming with toxigenicity.

The molecular mechanisms by which a stem cell is committed to individual lineage are largely unknown. Two different models, though not mutually exclusive, are currently debated. The first describes the temporal and hierarchical coordination of lineage-specific transcriptional programs. The second suggests that multilineage genes are expressed in a self-renewing and undifferentiated cell prior to lineage commitment. To challenge these two models in in vivo-appropriate conditions, the expression of an exogenous toxigene was used to create transgenic animals in which an inducible, reversible cell knock-out at a specific stage of differentiation could be achieved. Both additional transgenesis using the megakaryocyte specific alphaIIb promoter and targeted transgenesis were used to express the herpes virus thymidine kinase (tk) gene in the megakaryocytic lineage. When the tk gene was targeted to the locus of the megakaryocyte-specific alphaIIb gene, a typical Glanzman thrombasthenic syndrome was created. Despite this bleeding disorder, the lack of expression of the alphaIIb gene did not affect the development of the mice. In both transgenic and targeted animals, all progenitor cells were sensitive to the effect of the gancyclovir (GCV), both in vivo and ex vivo. Long-term bone marrow cell cultures on stromal layers indicated that most of the very early progenitor cells expressed the enzyme. All the results obtained with this inducible toxic phenotype indicated that genetic programs that are in control of the expression of lineage-specific genes are operative in a totipotent stem cell prior to lineage commitment and strongly support the concept that stem cells express a multilineage transcriptome.

A 2.7-kb portion of the 5' flanking region of the murine glycoprotein alphaIIb gene is transcriptionally active in primitive hematopoietic progenitor cells.

The continuous generation of mature blood cells from primitive multipotent progenitor cells requires a highly complex series of cellular events that are still largely unknown. To examine the molecular events associated with the commitment of these hematopoietic progenitor cells to the megakaryocytic lineage, the alpha subunit of the platelet integrin alphaIIb beta3 was used as marker. Despite an abundance of information regarding the role of this integrin in platelet adhesion and aggregation, the mechanisms that control the expression of the genes that code for these proteins are poorly understood and the earliest hematopoietic cell capable of expressing them has not been clearly identified. Thus, a strategy was developed to eradicate, using a conditional toxigene, all the hematopoietic cells capable of expressing the alphaIIb gene in mice. This was achieved by targeting the expression of the gene encoding the herpes simplex virus thymidine kinase (tk), specifically to these cell types, using a 2.7-kb fragment of the 5'-flanking region of the murine alphaIIb gene. Three transgenic lines having 1, 3, and 4 copies of the transgene, respectively were produced and analyzed. Administration of ganciclovir (GCV) to these mice induced a severe thrombocytopenia, which was due to the depletion of the entire megakaryocytic lineage, as shown by bone marrow (BM) culture and electron microscopy analysis. The time required to attain a severe thrombocytopenia was dependent on the level of the expression of the transgene and varied from 7 to 11 days. This condition was completely reversed when GCV treatment was discontinued. Progenitor cell assays showed that the alphaIIb promoter was active in primitive hematopoietic progenitor cells possessing myeloid, erythroid, and megakaryocytic potential and that the transcriptional activity of the promoter decreased progressively as differentiation proceeded towards the erythroid and myeloid lineages.

Dry mass allocation, water use efficiency and delta(13)C in clones of Eucalyptus grandis, E. grandis x camaldulensis and E. grandis x nitens grown under two irrigation regimes.

Clonal variation in water use efficiency (WUE), dry mass accumulation and allocation, and stable carbon isotope ratio (delta(13)C) of crude leaf fiber extracts was determined in six clones of Eucalyptus grandis W. Hill ex Maiden. grown for 16 months in field lysimeters in two soil water regimes. The relationships between delta(13)C and WUE calculated on the basis of leaf, harvestable stem, shoot and whole-plant dry mass accumulation were investigated. There was no clonal variation in dry mass accumulation but clonal allocation to roots, harvestable stems, branches and leaves varied. Water use efficiencies (mass of plant or plant part/water used over 16 months) differed significantly between clones. The clonal ranking of WUE varied depending on the units of dry mass accumulation used. Significant relationships between delta(13)C values and instantaneous water use efficiencies and ratios of internal leaf to ambient CO(2) concentrations were found only in the high soil water treatment. There were no relationships between delta(13)C values and whole-plant, shoot and harvestable stem water use efficiencies and soil water availability. Values of delta(13)C were negatively correlated with dry mass accumulation in the low soil water treatment. At the whole-plant level, WUE was positively correlated with dry mass accumulation in the high soil water treatment. We found significant differences in delta(13)C values between clones and the clonal rankings in delta(13)C and WUE were maintained in both soil water treatments.

Dissecting megakaryocytopoiesis in vivo with toxigenes.

The genetic programs that regulate the commitment of a totipotent stem cell to the megakaryocytic lineage remain poorly defined and require appropriate in vivo models. Using a cell-specific obliteration technique, a transgenic mouse model was produced where perturbations of megakaryocytopoiesis and platelet production may be induced on demand. This was achieved by targeting the expression of the herpes virus thymidine kinase (HSV-tk) to megakaryocytes using the regulatory regions of the gene coding for the alphaIIb gene, an early marker of megakaryocytopoiesis, which encodes the alpha subunit of the platelet integrin alphaIIb beta3. The HSV-tk gene is not toxic by itself, but sensitizes the target cell to the effect of ganciclovir (GCV), leading to the inhibition of DNA synthesis in dividing cells. The programmed eradication of the megakaryocytic lineage was induced by treating transgenic mice bearing the hybrid construct (alphaIIb-tk) with GCV. After 10 days of treatment, the platelet number was reduced by greater than 96.5% and megakaryocytes were not detectable in the bone marrow (BM). After discontinuing GCV, BM was repopulated with megakaryocytes, and the platelet count was restored within seven days. The recovery was accelerated by the administration of interleukin 11. Prolonged GCV treatment induced erythropenia in the transgenic mice. Assays of myeloid progenitor cells in vitro demonstrated that the transgene was expressed in early erythro-megakaryocytic bipotent progenitor cells. The reversibility and facility of this system provide a powerful model to determine both the critical events in megakaryocytic and erythroid lineage development, and for evaluating the precise role that platelets play in the pathogenesis of a number of vascular occlusive disorders.

Suppression of erythro-megakaryocytopoiesis and the induction of reversible thrombocytopenia in mice transgenic for the thymidine kinase gene targeted by the platelet glycoprotein alpha IIb promoter.

The mechanisms that regulate the commitment of a totipotent stem cell to the megakaryocytic lineage are largely unknown. Using a molecular approach to the study of megakaryocytopoiesis and platelet production, mice in which thrombocytopoiesis could be controlled were produced by targeting the expression of the herpes simplex virus thymidine kinase toxigene to megakaryocytes using the regulatory region of the gene encoding the alpha subunit of the platelet integrin alpha IIb beta 3. The programmed eradication of the megakaryocytic lineage was induced by treating transgenic mice bearing the hybrid construct (alpha IIbtk) with the antiherpetic drug ganciclovir (GCV). After 10 d of treatment, the platelet number was reduced by > 94.6%. After discontinuing GCV, the bone marrow was repopulated with megakaryocytes and the platelet count was restored within 7 d. Prolonged GCV treatment induced erythropenia in the transgenic mice. Assays of myeloid progenitor cells in vitro demonstrated that the transgene was expressed in early erythro-megakaryocytic progenitor cells. The reversibility and facility of this system provides a powerful model to determine both the critical events in megakaryocytic and erythroid lineage development and for evaluating the precise role that platelets play in the pathogenesis of a number of vascular occlusive disorders.

Three novel SMR1-related cDNAs characterized in the submaxillary gland of mice show extensive evolutionary divergence in the protein coding region.

We have previously characterized an abundant male-specific mRNA from the submaxillary gland (SMG) of rats, encoding the SMR1 (androgen-regulated) protein, which has the structure of a prohormone and is processed by maturation enzymes to release a small peptide in the blood and saliva. We have now characterized three SMR1-related cDNAs in the SMG of Balb/c mice. These cDNAs encode three novel proteins, designated MSG1, MSG2 and MSG3. They are 639, 662 and 471 nucleotides (nt) long, respectively, and the corresponding mRNAs appear to be expressed only in the SMG. The putative polypeptides they encode carry an N-terminal secretory peptide sequence and are, therefore, presumably secreted into saliva. Although closely related, the three mRNAs show striking differences: a particularly different expression pattern and an extremely high degree of variability observed in the central part of the molecules. The MSG1 and MSG3 cDNAs are identical, except for a 173-bp insert found only in MSG1. This insert contains three Pro-rich repeats (GPGIGRPPPPPP), reminiscent of the most abundant multigenic family of the SMG, the Pro-rich proteins (PRP). Although MSG1 shares several common features with PRP, it is structurally related to SMR1. The unusually high ratio of replacement/silent nt changes provides a basis to address complex aspects concerning the molecular events leading to the emergence of new proteins in the SMG.

Regrowth and tannin production in woody and succulent karoo shrubs in response to simulated browsing.

Allocation of carbon to chemical defences has often been suggested to be a direct response to browsing or grazing by herbivores. This study quantitatively compares total polyphenol and tannin production in response to simulated browsing of three karoo shrubs in order to test this induced defence hypothesis. The three species studied responded to browsing either by rapid regrowth or by increasing polyphenol production in the remaining tissues. The patterns did not follow any phylogenetic relationships but were weakly associated with the palatability of each species. The highly palatable deciduous species Osteospermum sinuatum, which is capable of rapid regrowth, showed no or very low levels of constitutive and browsing-induced total polyphenols, condensed tannins and protein-precipitating tannins. The evergreen sclerophyllous species Pteronia pallens showed a limited regrowth capacity and had intermediate levels of polyphenols, while the evergreen succulent species Ruschia spinosa showed almost no regrowth over the study period. R. spinosa contained the highest constitutive and browsing-induced levels of polyphenols, condensed tannins and protein-precipitating tannins. In two of the species more than one anti-herbivore defence feature co-occur. P. pallens foliage contains both hepatotoxins and polyphenols while R. spinosa has both structural (spines) and chemical defences. Responses of karoo shrubs to simulated browsing are interpreted as the result of passive alterations in plant chemistry rather than as an active defence response to herbivores.