Hydrolysis of nitriles by soil bacteria: variation with soil origin.

AIMS: The aim of this study was to explore bacterial soil diversity for nitrile biocatalysts, in particular, those for hydrolysis of ß-substituted nitriles, to the corresponding carboxamides and acids that may be incorporated into peptidomimetics. To achieve this, we needed to compare the efficiency of isolation methods and determine the influence of land use and geographical origin of the soil sample. METHODS AND RESULTS: Nitrile-utilizing bacteria were isolated from various soil environments across a 1000 km long transect of South Africa, including agricultural soil, a gold mine tailing dam and uncultivated soil. The substrate profile of these isolates was determined through element-limited growth studies on seven different aliphatic or aromatic nitriles. A subset of these organisms expressing broad substrate ranges was evaluated for their ability to hydrolyse ß-substituted nitriles (3-amino-3-phenylpropionitrile and 3-hydroxy-4-phenoxybutyronitrile) and the active organisms were found to be Rhodococcus erythropolis from uncultivated soil and Rhodococcus rhodochrous from agricultural soils. CONCLUSIONS: The capacity for hydrolysis of ß-substituted nitriles appears to reside almost exclusively in Rhodococci. Land use has a much greater effect on the biocatalysis substrate profile than geographical location. SIGNIFICANCE AND IMPACT OF THE STUDY: Enzymes are typically substrate specific in their catalytic reactions, and this means that a wide diversity of enzymes is required to provide a comprehensive biocatalysis toolbox. This paper shows that the microbial diversity of nitrile hydrolysis activity can be targeted according to land utilization. Nitrile biocatalysis is a green chemical method for the enzymatic production of amides and carboxylic acids that has industrial applications, such as in the synthesis of acrylamide and nicotinamide. The biocatalysts discovered in this study may be applied to the synthesis of peptidomimetics which are an important class of therapeutic compounds.

Characterisation of two bifunctional cellulase-xylanase enzymes isolated from a bovine rumen metagenome library.

Ruminant digestive tract microbes hydrolyse plant biomass, and the application of metagenomic techniques can provide good coverage of their glycosyl hydrolase enzymes. A metagenomic library of circa 70,000 fosmids was constructed from bacterial DNA isolated from bovine rumen and subsequently screened for cellulose hydrolysing activities on a CMC agar medium. Two clones were selected based on large clearance zones on the CMC agar plates. Following nucleotide sequencing, translational analysis and homology searches, two cellulase encoding genes (cel5A and cel5B) belonging to the glycosyl hydrolyse family 5 were identified. Both genes encoded pre-proteins of about 62 kDa, containing signal leader peptides which could be cleaved to form mature proteins of about 60 kDa. Biochemical characterisation revealed that both enzymes showed alkaline pH optima of 9.0 and the temperature optima of 65 °C. Substrate specificity profiling of the two enzymes using 1,4-ß-D-cello- and xylo-oligosaccharides revealed preference for longer oligosaccharides (n ≥ 3) for both enzymes, suggesting that they are endo-cellulases/xylanases. The bifunctional properties of the two identified enzymes render them potentially useful in degrading the ß-1,4 bonds of both the cellulose and hemicellulose polymers.

Self-reported beta-lactam allergy in government and private hospitals in Cape Town, South Africa.

BACKGROUND: Up to a quarter of inpatients in high-income countries (HICs) self-report beta-lactam allergy (BLA), which if incorrect,increases the use of alternative antibiotics, worsening individual health outcomes and driving bacterial resistance. In HICs, up to 95% ofself-reported BLAs are incorrect. The epidemiology of BLA in low- and middle-income African countries is unknown. OBJECTIVES: To describe the epidemiology and de-labelling outcomes of self-reported BLA in hospitalised South African (SA) patients. METHODS: Point-prevalence surveys were conducted at seven hospitals (adult, paediatric, government and privately funded, district andtertiary level) in Cape Town, SA, between April 2019 and June 2021. Ward prescription records and in-person interviews were conductedto identify and risk-stratify BLA patients using the validated PEN-FAST tool. De-labelling was attempted at the tertiary allergy clinic atGroote Schuur Hospital. RESULTS: A total of 1 486 hospital inpatients were surveyed (1 166 adults and 320 children). Only 48 patients (3.2%) self-reported a BLA,with a higher rate in private than in government-funded hospitals (6.3% v. 2.8%; p=0.014). Using the PEN-FAST tool, only 10.4% (n=5/48)of self-reported BLA patients were classified as high risk for true penicillin hypersensitivity. Antibiotics were prescribed to 70.8% (n=34/48)of self-reported BLA patients, with 64.7% (n=22/34) receiving a beta-lactam. Despite three attempts to contact patients for de-labelling atthe allergy clinic, only 3/36 underwent in vivo testing, with no positive results, and 1 patient proceeded to a negative oral challenge. CONCLUSION: Unlike HICs, self-reported BLA is low among inpatients in SA. The majority of those who self-reported BLA were low risk fortype 1 hypersensitivity, but outpatient de-labelling efforts were largely unsuccessful.

In vitro toxicity and internalization of gold nanoparticles (AuNPs) in human epithelial colorectal adenocarcinoma (Caco-2) cells and the human skin keratinocyte (HaCaT) cells.

Ingestion and transdermal delivery are two common routes of nanoparticle (NP) exposure. In this study, the intracellular uptake, cytotoxicity and genotoxicity of 14 nm and 20 nm citrate-stabilized gold nanoparticles (AuNPs), 14 nm polyethylene glycol (PEG)-liganded carboxyl AuNPs, 14 nm PEG-liganded hydroxyl AuNPs and 14 nm PEG-liganded amine AuNPs were assessed on human epithelial colorectal adenocarcinoma (Caco-2) cells and the human skin keratinocyte (HaCaT) cells. The uptake of AuNPs in the cells was confirmed through darkfield microscopy and hyperspectral imaging followed by spectral angle mapping (SAM). A high level of citrate AuNPs was found in both cell lines whilst uptake of PEGylated AuNPs was low, irrespective of their functional groups. Cytotoxicity assessed by cell impedance was only observed for the 14 nm citrate-stabilized AuNPs. Enhanced cell proliferation was also observed in 14 nm PEG-liganded hydroxyl and 14 nm PEG-liganded amine AuNP-treated Caco-2 and HaCaT cells. For the assessment of genotoxicity, the in vitro micronucleus assay was used. Dose-dependent genotoxicity was observed in both Caco-2 and HaCaT cells, with all the AuNPs inducing genotoxicity. In conclusion, the entry of NPs into the cells as well as toxicity was dependent on their physicochemical properties such as surface coating and different chemical functional groups.

The culturable seed mycobiome of two Banksia species is dominated by latent saprotrophic and multi-trophic fungi.

Seed fungal endophytes play an important beneficial role in the formation of the seedling mycobiome and contribute to plant establishment, but can also occur as latent pathogens and saprotrophs. Current knowledge on the function and diversity of seed fungal endophytes has been gained through studies in agricultural systems whilst knowledge from natural systems is relatively less. We used two co-occurring species from the genus Banksia from four sites in Australia's Sydney Basin Bioregion to investigate the abundance and diversity of seed fungal endophyte communities present in natural ecosystem hosts. Based on results from culturing and DNA sequence analysis of multiple loci, we found that Banksia seeds house a diverse range of fungal endophyte species, that when assigned to functional guilds belonged to multiple trophic modes. Thirty-one of the fungal taxa identified had not been previously reported as endophytes. Amongst the 58 Operational Taxonomic Units identified, Leotiomycetes and Sordariomycetes were the dominant classes and Banksiamyces (Leotiomycetes) and Penicillium (Sordariomycetes) the dominant genera, with many of the species isolated recorded in the literature as having a limited distribution. The two Banksias shared few fungal endophyte species, which were not always present across all study sites. We revealed a 'hidden diversity' within seeds of Banksia from natural ecosystems and provided insights into the influence host species can have on the seed mycobiome.

Application of European standards for health and quality control of game meat on game ranches in South Africa.

The health and quality compliance of game carcasses (n = 295) intended for the South African export market and aspiring to comply with the strict hygiene requirements of the European Union were compared with game carcasses (n = 330) available for the local market and currently not subjected to meat safety legislation. Samples were collected in similar seasons and geographical areas in South Africa from 2006 to 2009. Aerobic plate counts (APC) of the heart blood verified that both groups possessed similar ante mortem bacterial status. For health compliance APC, tests for Escherichia coli, Salmonella spp. and Staphylococcus aureus were performed on the carcasses. Surfaces of the local carcasses were swabbed using the European Enviro-biotrace sponge technique at 3 and 72 h post mortem. Unskinned but eviscerated export carcasses in the abattoir were skinned and sampled by incision using a corkborer 72 h post mortem. Temperature and pH readings were recorded at 3 and 72 h post mortem from the longissimus dorsi muscle and the readings at 3 h differed (P = 0.035). Temperatures at 72 h were lower for export than local carcasses (P < 0.001) because of earlier introduction and maintenance of the cold chain. The pH readings also differed between groups at 3 and 72 h (P < 0.001). APC results for the local group exceeded the maximum permissible count (< 10(5)). S. aureus results showed differences (P < 0.001), with readings from the local group being higher. The same tendency was exhibited for E. coli (P = 0.008). Imposition of hygiene guidelines for game ranchers producing meat for the local market is therefore recommended.

Enhancing the survival and expansion potential of a founder population through clonality.

• Although founder populations often have low diversity, they can potentially serve as stepping stones for further colonization, as refugia during nonoptimal times and as a source of specialized adaptive potential. The demonstration of such potential within natural plant populations has proven to be particularly difficult. Our investigation into a geographically disjunct population of a heterostylous shrub, Erythroxylum pusillum, aims to explore the evolutionary and ecological consequences of being an isolated founder population. • Microsatellite-based analyses were used to find evidence for, and trace the origins of, a severe founder effect. Molecular and spatial evidence was used to quantify clonality and to discover proof of somaclonal mutations. • We describe the unprecedented case of an isolated population that persisted through historical environmental fluctuations and in marginal habitat through vegetative spread, and is counteracting the lack of sexual recombination and gene flow through somatic mutation. • Our findings advance our understanding of how founder populations survive, differentiate and evolve. They also have implications for how conservation agencies should perceive and manage previously considered 'dead-end' populations.

An investigation of the effects of secondary processing on Mycobacterium spp. in naturally infected game meat and organs.

The risk for humans to contract bovine tuberculosis through the consumption of undercooked game meat as well as biltong (traditionally dried game meat) is a concern. The survival potential of Mycobacterium bovis during the cooking and drying processes was researched in a preceding study on beef and the positive results compelled the authors to investigate the results with a similar preliminary study on game meat. Muscular, lymphatic and visceral tissues from skin test positive African buffalo (Syncerus caffer) and greater kudu (Tragelaphus strepsiceros) with tuberculous lesions were collected from the Hluhluwe iMfolozi Park during the park's culling programme. The different tissues were exposed to cooking and the muscular tissue to the drying process prior to culture. All acid-fast isolates were analysed by polymerase chain reaction for the presence of Mycobacterium bovis. All tissues were found negative for Mycobacterium bovis but non-tuberculous mycobacteria were isolated from kidney, liver, heart and lymph nodes. The results showed that these processes will kill Mycobacterium bovis but the unexpected recovery of non-tuberculous mycobacteria suggests possible survival and resistance characteristics of these strains which might be of veterinary public health interest.

Cooking and drying as effective mechanisms in limiting the zoonotic effect of Mycobacterium bovis in beef.

For this study 48 non-infected muscle, lymphatic and visceral bovine tissue samples were collected from an approved red meat abattoir and spiked with 8 x 10(7) cfu/ml of M. bovis. The different spiked samples were subjected to cooking and drying (drying through the process of biltong-making) processes in a controlled laboratory environment. Mycobacterial isolates confirmed as M. bovis by means of polymerase chain reaction (PCR) were observed in 17 of a total of 576 samples that were exposed to the secondary processing method of cooking. The study showed that not only can M. bovis survive the cooking process but the survival of the bacterium will be determined by its unique adaptive changes to the surrounding composition of the environment. The results for the samples exposed to the drying process (n = 96) did not show any growth, suggesting that the process of biltong production as used in this study is likely to render infected meat safe for human consumption.

The effect of antibiotic timing on culture yield in paediatric osteoarticular infection.

PURPOSE: To assess the influence of antibiotic timing on surgical culture yield in paediatric patients with haematogenous osteoarticular infection. METHODS: All patients aged 0 to 15 years admitted to a National Children's Hospital with the diagnosis of acute, haematogenous, osteoarticular infection (osteomyelitis and/or septic arthritis) between June 1997 and December 2016 were retrospectively analyzed. Only patients with positive blood cultures undergoing surgery for culture and debridement were included. Patients were allocated into pre-treatment and post-treatment groups, according to whether they received antibiotics before or after surgical cultures were obtained. Outcomes measured included baseline variables, treatment characteristics and surgical culture yield. RESULTS: A total of 131 patients were included; 107 patients in the pre-treatment group and 24 patients in the post-treatment group. There was no significant difference with respect to patient age (p = 0.870), white blood cell count (p = 0.197), ethnicity (p = 0.203) or infection multi-focality (p = 0.883) between the two groups.The administration of systemic antibiotics prior to obtaining surgical cultures had no clinically significant effect on surgical culture yield (rate of positive surgical cultures, 85% (pre-treatment) versus 54.2% (post-treatment); p = 0.002). Within the pre-treatment group, there was no significant difference in duration of pre-surgical antibiotic treatment between patients who had positive or negative surgical cultures (mean duration, 45.9 hours (positive cultures) versus 47.9 hours (negative cultures); p = 0.743). CONCLUSION: In paediatric patients with acute, haematogenous, osteoarticular infection, antibiotic administration before surgery does not decrease surgical culture yield. Our results suggest that paediatric patients presenting with suspected osteoarticular infection should receive appropriate systemic antibiotics promptly after blood cultures are obtained. LEVEL OF EVIDENCE: Level III - retrospective case-control study.

Dehydro-brachylaenolide: an eudesmane-type sesquiterpene lactone.

The three-ring eudesmanolide, C(15)H(16)O(3), is a natural product isolated from Dicoma anomala Sond. (Asteraceae). The compound contains an endo-exo cross conjugated methyl-enecyclo-hexenone ring with an envelope conformation trans-fused with cyclo-hexane and trans-annelated with an α-methyl-ene γ-lactone. The absolute structure was assigned by optical rotation measurements compared to those from the synthetic compound with known stereochemistry. The crystal packing is consolidated by C-H⋯O interactions.

p47phox is required for atherosclerotic lesion progression in ApoE(-/-) mice.

NADPH oxidase is upregulated in smooth muscle cells (SMCs) in response to growth factor stimulation, concomitant with increased reactive oxygen species (ROS) production. We investigated the role of ROS production by NADPH oxidase in SMC responses to growth factors and in atherosclerotic lesion formation in ApoE(-/-) mice. SMCs from wild-type, p47phox(-/-), and gp91phox(-/-) mice differed markedly with respect to growth factor responsiveness and ROS generation. p47phox(-/-) SMCs had diminished superoxide production and a decreased proliferative response to growth factors compared with wild-type cells, whereas the response of gp91phox(-/-) SMCs was indistinguishable from that of wild-type SMCs. The relevance of these in vitro observations was tested by measuring atherosclerotic lesion formation in genetically modified (wild-type, p47phox(-/-), ApoE(-/-), and ApoE(-/-)/p47phox(-/-)) mice. ApoE(-/-)/p47phox(-/-) mice had less total lesion area than ApoE(-/-) mice, regardless of whether mice were fed standard chow or a high-fat diet. Together, these studies provide convincing support for the hypothesis that superoxide generation in general, and NADPH oxidase in particular, have a requisite role in atherosclerotic lesion formation, and they provide a rationale for further studies to dissect the contributions of ROS to vascular lesion formation.

Obesity in South Africa.

South Africa has not been spared in the rampant global increase in obesity. Throughout Africa, as elsewhere, ethnicity has a major impact on the incidence and pathogenesis of comorbid diseases, particularly diabetes. Combined figures for obesity and overweight (body mass index [BMI] > 25 kg m(-2)) obtained across all ethnic groups in the adult population in 1998, were 57% for women and 29% for men. From the 1960s until the late 1980s, the notion of 'healthy' or 'benign' obesity was propagated in South Africa. Not surprisingly, this led to ignorance around the problem of obesity, and treatment of some of the comorbid diseases was neglected. Fortunately, as an increasing number of seminal studies draw us closer to reality, the misperception of benign obesity is being corrected. This is allowing us to address the real issues underlying the current epidemic, and to recognize and manage the comorbid diseases, in particular type 2 diabetes. A new framework for research is also emerging as we begin to define the factors underlying the impact of ethnicity on obesity.

Professional nurses' attitudes towards providing termination of pregnancy services in a tertiary hospital in the north west province of South Africa.

The Choice on Termination of Pregnancy Act (no 92 of 1996) was implemented during 1997. This study attempted to investigate professional nurses' attitudes towards rendering termination of pregnancy (TOP) services at a tertiary hospital in the North West Province of South Africa. A quantitative descriptive research design was used to study professional nurses' attitudes towards providing TOP services. The research results, obtained from questionnaires completed by professional nurses, indicated that most professional nurses' attitudes included that women should be at least 16 years of age to access these services; women should not be able to access repeated TOPs; nurses would prefer to administer pills rather than to use vacuum aspirations; nurses should work in TOP services by choice only. TOP centers should have better equipment, more resources and more staff members. Nurses working in TOP services would appreciate receiving more support from their families, friends, managers and communities. Some professional nurses experienced guilt, depression, anxiety and religious conflicts as a result of providing TOP services. Despite the legalisation of TOPs, these services remained stigmatised. Professional nurses did not want to work in these services and also did not want to be associated with them.

A study of ignition of metal impregnated carbons: the influence of oxygen content in the activated carbon matrix.

A study of the reason for the early ignition of coconut-based impregnated carbon in comparison with the peat-based impregnated carbon was conducted. The surface features of carbons were evaluated using various physicochemical methods. The metal analysis of the initial carbon indicated that the content of potassium was higher in the coconut-based carbon. The surface functional group analysis revealed the presence of similar surface species; however, the peat-based carbon was more acidic in its chemical nature. Since the oxygen content was higher in the peat-based carbon, the early ignition of the coconut-based material was attributed to its higher affinity to chemisorb oxygen, which leads to exothermic effects. This conclusion was confirmed by performing oxidation of coconut-based carbon prior to impregnation. This process increased the ignition temperature for Cu/Cr impregnated coconut-based material from 186 to 289 degrees C and for the Cu/Zn/Mo impregnated carbon from 235 to 324 degrees C.

Nurses' perceptions of their psychiatric nursing competencies in the Kwa-Zulu Natal Province.

This research report discusses the results of a quantitative exploratory descriptive survey which investigated specific psychiatric competencies of nurses who completed the R425 diploma programme and who were working in psychiatric clinical units in and around Durban and Pietermaritzburg during 2003. Forty-five nurses completed questionnaires, indicating their perceptions of their psychiatric nursing competencies on Likert scales and by responding to open-ended questions. The Statistical Package for Social Sciences, (SPSS) was used to analyse the data from the 45 completed questionnaires. Findings revealed that nurses who completed the R425 programme, and who were working in psychiatric clinical units, perceived themselves to be competent in performing some of the psychiatric clinical competencies contained in the questionnaires, but not all.

Analysis of co-transfected expression vectors amplified by methotrexate selection in rat-1 cells.

In an earlier investigation of the influence of high level expression of p21H-ras, rat-1 cells were co-transfected with a selectable vector (pSV2Neo), an amplifiable vector (encoding dihydrofolate reductase; DHFR) and an H-ras expression vector. In this study we have analyzed the gene dose and expression levels of the three co-transfected plasmid vectors in cell lines that had been selected and isolated at different methotrexate concentrations. Growth of the cells in the absence of selection and Southern blot analyses indicate that the transfected vectors are stably co-integrated into the host genome. High expression levels from all three co-transfected vectors were evident at both the mRNA and protein levels, indicating that they are tightly linked in the host genome. The presence of a large amount of unspliced H-ras mRNA in cells expressing high levels of H-ras p21 indicates that processing of mRNA may be rate-limiting. Comparison of the gene dose and expression levels shows that the resistance of cells to increased methotrexate concentrations can occur by different mechanisms. It is concluded that co-transfection of individual plasmid vectors into rat-1 cells, followed by methotrexate selection, is an effective manner of achieving high level expression of proteins in cultured cells.

Enzymatic conversion of the major polypeptide chains of thyroglobulin.

The subunit composition of fully reduced bovine thyroglobulin consists of two polypeptide chains with molecular weights near 300,000. These two chains have been separated on sodium dodecyl sulfate gels by electrophoresis and were referred to as S and F. The effect of incubating native bovine thyroglobulin with a commercial preparation of horseradish peroxidase resulted in the conversion of S to F (and faster migrating polypeptides). Since this reaction was independent of the presence of H2O2 and iodide, it was necessary to demonstrate that the activity was present in the peroxidase enzyme which could be identified by its heme group. When the enzyme preparation was fractionated on Sephadex G-100, the activity responsible for the conversion of S to F was not associated with the heme peak but with another protein peak which eluted later on the column. Proteolytic inhibitors, such as phenylmethyl sulfonyl fluoride and pancreatic trypsin inhibitor, caused partial inhibition of the activity responsible for the conversion of S to F, whereas inhibitors of peroxidase activity had no effect. Guinea pig thyroglobulin was less susceptible to the proteolytic activity present in the peroxidase preparation than bovine thyroglobulin. The subunit composition of guinea pig thyroglobulin is different from that of bovine, since three bands of different sizes are present. The 300,000 mol wt subunit of guinea pig thyroglobulin (band A) ws largely degraded, whereas the 210,000 mol wt subunit (band B) was partially degraded and the 100,000 mol wt subunit (band C) was unaffected.

Lactate and glycerol release from adipose tissue in lean, obese, and diabetic women from South Africa.

Abnormalities observed in intermediary metabolism may be related to the pathogenesis of obesity-related diseases such as type 2 diabetes. Glycerol and lactate production was estimated in the sc adipose tissue of two anatomical regions of 10 lean (LW), 10 obese (OW), and 10 matched diabetic (DW) black urban women. This was done with the sc microdialysis technique and combined with adipose tissue blood flow (ATBF) rates calculated from (133)Xe clearance. Biochemical measurements were made in the postabsorptive and postprandial state. Bioimpedance and computed tomography scans were used to define body composition. DW present with more visceral fat (DW, 138 +/- 5.0; OW, 66.6 +/- 5.0 cm; P < 0.01). This was associated with elevated free testosterone levels (DW, 1.21 +/- 0.1; OW, 0.75 +/- 0.1 nmol/L; P < 0.05). The fasting FFA, glycerol, and lactate levels increased across the three groups (LW < OW < DW). During the oral glucose tolerance test, glucose levels were elevated in DW, with higher insulin levels [0 h: DW, 207 +/- 8.6; OW, 100 +/- 7.2 pmol/L (P < 0.01); 1 h: DW, 410 +/- 15.2; OW, 320 +/- 10.9 pmol/L (P < 0.05)], but with a flat Cpeptide response (1 h: DW, 932 +/- 40; OW, 1764 +/- 40 pmol/L; P < 0.05). Plasma lactate levels increased significantly in LW and OW at 1 h (P < 0.001), but remained lower in LW vs. OW for all time points. ATBF was highest in LW [abdominal, 0 h: DW, 4.5 +/- 0.2; OW, 1.7 mL/100 g.min (P < 0.01); femoral, 0 h: DW, 3.4 +/- 0.2; OW, 1.8 +/- 0.3 mL/100 g.min (P < 0.01)]. ATBF did not increase in DW during the oral glucose tolerance test. Glycerol release (GR) was used to assess the lipolytic rate and was highest in LW in the abdominal area [0 h: LW, 1.7 +/- 0.2; OW, 1.1 +/- 0.2 micromol/kg.min (P < 0.05); DW, 0.78 +/- 0.05 micromol/kg.min (P < 0.05 vs. OW)]. By contrast, GR was higher in the femoral area of OW (0 h: OW, 1.6 +/- 0.2; LW, 1.15 +/- 0.1 micromol/kg.min; P < 0.05). Regional differences were observed for GR in both OW and DW (femoral > abdominal). Lactate release (LR) was low in DW [abdominal, 0 h: DW, 3.5 +/- 0.4; OW, 7.8 +/- 1.0 micromol/kg.min (P < 0.001); femoral, 0 h: DW, 3.1 +/- 0.3; OW, 9.0 +/- 0.9 micromol/kg.min (P < 0.001)]. LR was appropriately low for body fat mass in LW, with a brisk increase between 0 and 1.5 h. A negative correlation exists between GR (abdominal area) and insulin levels in the postabsorptive state (P < 0.0001). In conclusion, 1) the fasting lipolytic rate is associated with insulin levels; 2) OW and DW have more adipose tissue insulin resistance than LW; 3) OW and DW have a brisker lipolysis in the femoral area; and 4) in DW, higher visceral mass is associated with elevated free testosterone and FFA concentrations. Obesity in the black population is therefore characterized by a marked degree of adipose tissue lipolysis. This degree of resistance together with increasing body fat mass may predispose the obese women to developing type 2 diabetes. Once this disease is established, the onset of adipose tissue vascular insulin resistance will sustain ongoing insulin resistance, even in the presence of relative insulinopenia.