The Kenyan Luo migration study: observations on the initiation of a rise in blood pressure.

OBJECTIVE--To demonstrate the magnitude, timing, and cause of changes in blood pressure that occur in migrants from a low blood pressure population on moving to an urban area. DESIGN--A controlled longitudinal observational study of migrants as soon after migration as possible and follow up at three, six, 12, 18, and 24 months after migration. A cohort of controls living in a rural area who were matched for age, sex, and locality were also observed at the same periods. SETTING--35 Villages on the northern shores of Lake Victoria in western Kenya and Nairobi. PARTICIPANTS--325 Members of the Luo tribe aged 15 to 34 years who had migrated to Nairobi and 267 controls living in villages. The numbers of both groups reduced during follow up such that only 63 migrants and 143 controls were followed up for two years. MAIN OUTCOME MEASURES--A medical questionnaire and three 24 hour diet histories were completed by migrants and controls. Height, weight, pulse, and blood pressure were measured. Three 12 hour overnight urine samples were collected from all participants and analysed for sodium, potassium, and creatinine concentrations. RESULTS--The mean systolic blood pressure of migrants was significantly higher than that of controls throughout the study, and the distribution of blood pressure was shifted to the right compared with controls. The mean diastolic blood pressure of the two groups diverged over time. Blood pressure differences were not due to selective migration. The migrants'' mean urinary sodium:potassium ratio was higher than that of controls (p less than 0.001) throughout, and weight and pulse rate were also higher among migrants, although differences diminished with time. CONCLUSIONS--Urinary sodium:potassium ratio, pulse rate, and weight are important predictors of increased blood pressure among migrants from a low blood pressure community and may also be implicated in the initiation of essential hypertension.     

The study of the process of fluid-phase endocytosis in cervical squamous cells using fluorescent microspheres

Physiological processes in cervical squamous epithelium have not been extensively studied. Perhaps understandably, most of the research has concentrated on the pathology of the cervix, in particular dysplasia and malignancy. Fluid-phase endocytosis is a physiological process which has been demonstrated to be important in understanding disease development at other squamous epithelial sites, e.g. oesophagus. In this study, we have demonstrated by a new methodology developed in our laboratory using fluorescent microspheres and flow cytometry that fluid-phase endocytosis occurs in cervical squamous cells. The process has been shown to be dose- and time-dependent. This novel approach provides a means to improve our understanding of the physiological functions of the cervix and may provide insight into the pathogenesis of cervical neoplastic and non-neoplastic disease.     

The effect of macromolecules upon the rates of hydrolysis of aromatic nitrogen mustard derivatives

The rates of hydrolysis of several aromatic nitrogen mustards (chlorambucil, melphalan, melphalan ester, and “p-phenylenediamine mustard”) have been determined in the presence of proteins and of synthetic and semisynthetic polymeric substances. Only those molecules with extensive hydrophobic areas significantly slowed down the hydrolysis of the alkylating agents. A substance able to protect a nitrogen mustard against hydrolysis does not necessarily affect the biological activity of the agent when combined with it.     

Electron spin resonance studies of changes in membrane fluidity of Chinese hamster ovary cells during the cell cycle

Electron spin resonance (ESR) spin-label methods were used with 5-doxyl-stearic acid as a probe to investigate membrane fluidity of Chinese hamster ovary (CHO) cells during the cell cycle. A 35 GHz ESR technique was developed to study membrane fluidity of intact cells. This technique requires only about $\text{1}\text{6}$ the amount of cells compared to the conventional spin-label techniques. With this technique we observed a cyclic change of membrane fluidity during the cell cycle of CHO cells: cells in mitosis had the highest membrane fluidity, whereas cells in G₁ and early S phases had the lowest membrane fluidity.     

Common antigenicity for two glycosidases

Enzyme replacement therapy (ERT) has proven to be an effective therapy for some lysosomal storage disorder (LSD) patients. A potential complication during ERT is the generation of an immune response against the replacement protein. We have investigated the antigenicity of two distantly related glycosidases, alpha-glucosidase (Pompe disease or glycogen storage disease type II, GSD II), and alpha-L-iduronidase (Hurler syndrome, mucopolysaccharidosis type I, MPS I). The linear sequence epitope reactivity of affinity purified polyclonal antibodies to recombinant human alpha-glucosidase and alpha-L-iduronidase was defined, to both glycosidases. The polyclonal antibodies exhibited some cross-reactive epitopes on the two proteins. Moreover, a monoclonal antibody to the active site of alpha-glucosidase showed cross-reactivity with a catalytic structural element of alpha-L-iduronidase. In a previous study, in MPS I patients who developed an immune response to ERT, this same site on alpha-L-iduronidase was highly antigenic and the last to tolerise following repeated enzyme infusions. We conclude that glycosidases can exhibit cross-reactive epitopes, and infer that this may relate to common structural elements associated with their active sites.     

Advantages of using same species enzyme for replacement therapy in a feline model of mucopolysaccharidosis type VI

In a feline model of mucopolysaccharidosis type VI (MPS VI), recombinant feline N-acetylgalactosamine-4-sulfatase (rf4S) administered at a dose of 1 mg/kg of body weight, altered the clinical course of the disease in two affected cats treated from birth. After 170 days of therapy, both cats were physically indistinguishable from normal cats with the exception of mild corneal clouding. Feline N-acetylgalactosamine-4-sulfatase was effective in reducing urinary glycosaminoglycan levels and lysosomal storage in all cell types examined except for corneal keratocytes and cartilage chondrocytes. In addition, skeletal pathology was nearly normalized as assessed by radiographic evidence and bone morphometric analysis. Comparison of results with a previous study in which recombinant human 4S (rh4S) was used at an equivalent dose and one 5 times higher indicated that rf4S had a more pronounced effect on reducing pathology than the same dose of rh4S, and in some instances such as bone pathology and lysosomal storage in aorta smooth muscle cells, it was as good as, or better than, the higher dose of rh4S. We conclude that in the feline MPS VI model the use of native or same species enzyme for enzyme replacement therapy has significant benefits.     

Mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome): a Y210C mutation causes either altered protein handling or altered protein function of N-acetylgalactosamine 4-sulfatase at multiple points in the vacuolar network

The lysosomal hydrolase N-acetylgalactosamine 4-sulfatase (4-sulfatase) is required for the degradation of the glycosaminoglycan substrates dermatan and chondroitin sulfate. A 4-sulfatase deficiency results in the accumulation of undegraded substrate and causes the severe lysosomal storage disorder mucopolysaccharidosis type VI (MPS VI) or Maroteaux-Lamy syndrome. A wide variation in clinical severity is observed between MPS VI patients and reflects the number of different 4-sulfatase mutations that can cause the disorder. The most common 4-sulfatase mutation, Y210C, was detected in approximately 10% of MPS VI patients and has been associated with an attenuated clinical phenotype when compared to the archetypical form of MPS VI. To define the molecular defect caused by this mutation, Y210C 4-sulfatase was expressed in Chinese hamster ovary (CHO-K1) cells for protein and cell biological analysis. Biosynthetic studies revealed that Y210C 4-sulfatase was synthesized at a comparable molecular size and amount to wild-type 4-sulfatase, but there was evidence of delayed processing, traffic, and stability of the mutant protein. Thirty-three percent of the intracellular Y210C 4-sulfatase remained as a precursor form, for at least 8 h post labeling and was not processed to the mature lysosomal form. However, unlike other 4-sulfatase mutations causing MPS VI, a significant amount of Y210C 4-sulfatase escaped the endoplasmic reticulum and was either secreted from the expression cells or underwent delayed intracellular traffic. Sixty-seven percent of the intracellular Y210C 4-sulfatase was processed to the mature form (43, 8, and 7 kDa molecular mass forms) by a proteolytic processing step known to occur in endosomes-lysosomes. Treatment of Y210C CHO-K1 cells with the protein stabilizer glycerol resulted in increased amounts of Y210C 4-sulfatase in endosomes, which was eventually trafficked to the lysosome after a long, 24 h chase time. This demonstrated delayed traffic of Y210C 4-sulfatase to the lysosomal compartment. The endosomal Y210C 4-sulfatase had a low specific activity, suggesting that the mutant protein also had problems with stability. Treatment of Y210C CHO-K1 cells with the protease inhibitor ALLM resulted in an increased amount of mature Y210C 4-sulfatase localized in lysosomes, but this protein had a very low level of activity. This indicated that the mutant protein was being inactivated and degraded at an enhanced rate in the lysosomal compartment. Biochemical analysis of Y210C 4-sulfatase revealed a normal pH optimum for the mutant protein but demonstrated a reduced enzyme activity with time, also consistent with a protein stability problem. This study indicated that multiple subcellular and biochemical processes can contribute to the biogenesis of mutant protein and may in turn influence the clinical phenotype of a patient. In MPS VI patients with a Y210C allele, the composite effect of different stages of intracellular processing/handling and environment has been shown to cause a reduced level of Y210C 4-sulfatase protein and activity, resulting in an attenuated clinical phenotype.