Light and electron microscopic localization of the alpha 1-chain and the E1 and E8 domains of laminin-1 in mouse kidney using monoclonal antibodies to establish the orientation of laminin-1 within basement membranes.

To localize the different domains of the laminin-1 molecule in tissues and gain insight into their in vivo relevance, we raised rat anti-mouse monoclonal antibodies (MAbs) against the entire molecule. Then we tested eight of the 20 clones producing anti-laminin-1 MAbs to specify their reactivity towards the alpha 1-, beta 1-, and gamma 1-chains and the elastase-cleaved fragments of the laminin-1 molecule. We found three MAbs with high titers in ELISA that showed good reactivity in embedded tissue. One of these reacted specifically against the E1 fragment, one against the E8 fragment, and one MAb detected the alpha 1-chain of laminin-1 but not the beta 1- or gamma 1-chain. All three MAbs are useful for light immunohistochemical investigations on cryosections and on paraffin-embedded material, and for ultrastructural localization of laminin-1 in LR Gold-embedded mouse tissue. Antibody staining of the E1 and E8 domains of laminin-1 revealed distinct localization of the molecule in the proximal tubule basement membranes of mouse kidney. The short arms (E1) of the laminin-1 molecule are predominantly located in the lamina lucida and the long arms (E8) are oriented towards the lamina fibroreticularis. Therefore, both MAbs are useful for studies of the orientation of the laminin-1 molecule in basement membranes. The distal tubule basement membranes did not show any distinct pattern of laminin-1 distribution. In general, the distal tubules showed the strongest reactions over the entire width of the basement membrane for all three MAbs. In contrast, the proximal tubule basement membranes showed somewhat weaker reactivity but a distinct pattern of laminin-1 distribution, with the E1 fragments oriented towards the adjacent epithelial cell surface.

Carbohydrate metabolism in U.S. Navy submarine personnel.

One- and two-hour postprandial glucose and insulin values were measured in 58 submariners with 5 or more FBM patrols and in 58 non-submariners. An interrelationship between serum glucose and insulin for classifying defects in carbohydrate metabolism indicated that 55% of the submariners and 45% of the non-submariners exhibited some type of defect. Exercise appears to play an important role in the maintenance of normal carbohydrate metabolism in these subjects. Non-submariners reported engaging in significantly more exercise than did submariners. Negative correlation between exercise vs. 1 and 2-h glucose and 2-h insulin was significant for all subjects. An inverse relationship was observed between amount of exercise and the severity of carbohydrate metabolic defects in submariners and non-submariners as well as in normal weight and obese individuals. No statistical differences were found between the submariner and non-submariner groups with respect to age, % body fat, fatness (normal or obese), glycosylated hemoglobin, or family history of diabetes.

Blood volume responses in partially dehydrated subjects working in the cold.

The effects of high-salt diets, cold, and heavy exercise have been examined in 33 Marine Corps volunteers living and working in a cold chamber. Temperatures varied from -29 degrees C during working hours to -4 degrees C, simulating partly warmed shelters, at night. Dehydrated operational rations were eaten and fluid intakes were controlled. When quantities of water were consumed at or above the amounts determined in previous studies in this series to prevent symptoms of cold-induced dehydration with these diets, ie., 2.5-3.0 L/d with diets containing 24 g of NaCl/d or about 2.0 L/d with 8 g NaCl/d, blood volumes increased 5-10%, primarily as a result of plasma volume expansion. This finding apparently confirms the results of earlier workers who have noted plasma volume increases in subjects undergoing vigorous exercise training. In subjects receiving 0.5 L/d or more below recommended quantities of water, intravascular volume increases developed slowly and quite erratically during 5 d of exercise. Intravascular volume increases responded within 1-2 d when fluid levels became appropriate for either high- or low-salt intakes. Besides confirming recommendations concerning water needed with dehydrated diets under conditions of the experiment, the observation of intravascular fluid volume increases during exercise training--despite limiting fluid intakes--raises the question of whether there are any physiological advantages from this adaptive mechanism.

GABA or sodium-bromide-induced plasticity of neurites of mouse neuroblastoma cells in culture. A quantitative study.

Differentiated C1300 mouse neuroblastoma cells were treated with 10(-4)-10(6) M gamma-aminobutyric acid (GABA) and/or sodium bromide (NaBr) for 2 days and then fixed. Quantitative studies revealed an increase in the length and branching of the processes, as well as an increase in the number of cells when compared to the controls. It is suggested that the above changes contribute to the augmentation of specialized contacts between cells and processes as well as the further maturation of the primitive stages of synaptogenesis as discussed.

Modified growth cones in mouse neuroblastoma cultures; an electron microscopic study.

Growth cones of differentiated neuroblastoma cells in monolayer cultures were studied by electron microscopy. Morphological differentiation of the growth cone formation was induced by sodium bromide. Upon prolonged application of 10(-4) to 10(-5) M sodium bromide to the cultures, a peculiar or modified formation of the growth cone occurred. Growth cones lengthened gradually. The ultrastructure of the growth cone in contrast to the control was typified by a round to oval structure, midway being electron-dense and carrying laterally denser cytoplasmic protrusions. Bundles of microtubules, aggregates of many dense-cored vesicles, 70-150 nm in diameter, a few less electron-dense, as well as some agranular vesicles were present. Comparing the findings with previous ultrastructural accounts of growth cones of cultured ganglion cells or neuroblastoma cells, differences outnumbered similarities. The organization of the microtubule bundles and the abundance of dense-cored vesicles, sometimes extending distally was remarkable. The presence of an electron-dense substance, of unknown origin, extending laterally with the cytoplasmic protrusions has not been describe as yet.

A simple embedding technique for monolayer neuronal cultures grown in plastic flasks.

An in situ embedding technique for monolayer cultures for subsequent ultrastructural studies is described. Dehydration is carried out in graded ethanol series without propylene oxide, and Epon is used as a final embedding medium. The advantage of this method is that the plastic flask with the embedded material can be stripped off easily without affecting the monolayer culture and that the plastic does not dissolve in Epon.

Biochemical and hematologic profiles of 1000 submariners.

Control biochemical and hematologic data were gathered for 1017 healthy submariners who ranged in age from 19.5 to 43.5 years. Means, standard deviation, and frequency distribution are presented for 24 whole blood and serum variables and, where appropriate, for 11 urinary variables. After statistical separation of the effects of aging and length of submarine service, it has been determined that the following correlations were significant in this sample: neutrophil and leucocyte levels, serum cholesterol, and both fasting and postprandial glucose correlated positively with age; serum alkaline phosphatase levels and age correlated negatively. Age-corrected positive correlations were demonstrable between length of submarine service and both serum cholesterol content and alkaline phosphatase activity; after a loading test, glucose levels showed a negative relationship to length of submarine service. Split-sample correlation analyses verified these significant correlations, with the exception of the apparent rise in alkaline phosphatase activity with increasing length of submarine duty.

Cardiovascular risk factors in submariners.

A sizeable group of biochemical, hematologic and physiologic variables that significantly affect the prognosis for diseases of aging, especially for coronary heart disease (CHD), were studied in 1017 submariners. Skinfold thickness in these subjects was determined to be higher than in most other groups of men of similar age; the total level of body fat was within the range of high normality. Serum cholesterol levels, cigarette smoking, relative weight, and blood pressure appeared to be the factors most directly responsible for the extent of cardiovascular risk in this group of submariners. Though submariners as a group do not apparently have appreciably higher levels of CHD risk than other American men, there was a significant tendency for total risk to increase with length of submarine service as well as with age. A similar age-independent increase in serum cholesterol correlating with length of submarine service was reported earlier. Split-sample analyses support the reliability of the age-corrected correlations of CHD risk with time of submarine service. Although no attempt was made to prove a direct relationship between alcohol consumption or coffee drinking and cardiovascular risk, there were strong correlations noted. These factors, combined with serum cholesterol levels, cigarette smoking, and relative weight, deserve consideration as potentially modifiable CHD risks.

Calcium, magnesium, and phosphorus metabolism, and parathyroid-calcitonin function during prolonged exposure to elevated CO2 concentrations on submarines.

Studies of calcium and phosphorus metabolism and acid-base balance were carried out on three Fleet Ballistic Missile (FBM) submarines during prolonged exposure to elevated concentrations of CO2. The average CO2 concentration in the submarine atmosphere during patrols ranged from 0.85% to 1% CO2. In the three studies, in which 9--15 subjects participated, the urinary excretion of calcium and phosphate fell during the first three weeks to a level commensurate with a decrease in plasma calcium and increase in phosphorus. In the fourth week of one patrol, a marked increase was found in urinary calcium excretion, associated with a rise in blood PCO2 and bicarbonate. Urinary calcium excretion decreased again during the 5th to 8th week, with a secondary decrease in blood pH and plasma calcium. During the third patrol, the time course of acid-base changes corresponded well with that found during the second patrol. There was a trend toward an increase in plasma calcium between the fourth and fifth week commensurate with the transient rise in pH and bicarbonate. Plasma parathyroid and calcitonin hormone activities were measured in two patrols and no significant changes were found. Hydroxyproline excretion decreased in the three-week study and remained unchanged in the second patrol, which lasted 57 days. It is suggested that during prolonged exposure to low levels of CO2 (up to 1% CO2), calcium metabolism is controlled by the uptake and release of CO2 in the bones. The resulting phases in bone buffering, rather than renal regulation, determine acid-base balance.

Biochemistry and hematology at decompression sickness: a case report.

A 24-year-old hospital corpsman, a volunteer in a series of dry chamber air dives to a simulated pressure equivalent to 188 FSWG (57.3 MSWG), developed left knee pain shortly after standard decompression. A tentative diagnosis of decompression sickness was made and recompression therapy was initiated with alleviation of pain occurring at 60 FSWG (18.3 MSWG). A U.S. Navy Treatment Table "5 (oxygen breathing) regimen was then selected and completed uneventfully. The subject had been undergoing biomedical evaluation for several days prior to diving; thus, a clinically diagnosed case of dysbarism with subsequent treatment was available for study. This individual was then monitored for a 10-d period. The acute phase of decompression sickness was characterized by a marked shortening of clotting time and a thrombocytopenia with accompanying increased platelet aggregates. The recovery phase was categorized by a variety of hematological and bio-chemical changes. Hemodilution, an elevated megathrombocyte index, and a tendency toward eosinopenia were evident for most of the 10-d observation period. Other persistent alterations detected during this period included a relative hyperglycemia, depressed urine Na+/K+, and increased ketosteroid excretion. These observations indicate that abatement of pain after treatment of dysbarism can be followed by the onset of a variety of biochemical and hematological changes. Moreover, complete recovery may require upwards of 10 d.